Methods of enhancing stem cell differentiation into beta cells

ABSTRACT

Disclosed herein are compositions and methods of enhancing stem cell differentiation into beta cells with use of one or more epigenetic modification compounds. The present disclosure also relates to compositions and methods of sorting and enriching the differentiated beta cells. The present disclosure also relates to compositions and methods of irradiating cell population for reducing proliferation.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.17/010,346, filed on Sep. 2, 2020, which is a continuation ofInternational Patent Application No. PCT/US2019/020430, filed Mar. 1,2019, which claims the benefit of U.S. Provisional Application No.62/637,923, filed on Mar. 2, 2018, each of which is incorporated hereinby reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Nov. 10, 2022, isnamed 47380-718_302_SL.xml and is 22,906 bytes in size.

BACKGROUND OF THE DISCLOSURE

Deciphering the molecular mechanisms that direct islet cellregeneration, plasticity and function can improve and expand the β cellreplacement strategies for treating diabetes. The generation of stemcell derived β-cells can provide a potentially useful step toward thegeneration of islets and pancreatic organs. One of the rapidly growingdiseases that may be treatable by stem cell derived tissues is diabetes.Type 1 diabetes results from autoimmune destruction of β-cells in thepancreatic islet. Type 2 diabetes results from peripheral tissue insulinresistance and β-cell dysfunction. Diabetic patients, particularly thosesuffering from type 1 diabetes, can potentially be cured throughtransplantation of new β-cells. Patients transplanted with cadaverichuman islets can be made insulin independent for 5 years or longer viathis strategy, but this approach is limited because of the scarcity andquality of donor islets. The generation of an unlimited supply of humanβ-cells from stem cells can extend this therapy to millions of newpatients and can be an important test case for translating stem cellbiology into the clinic.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.Absent any indication otherwise, publications, patents, and patentapplications mentioned in this specification are incorporated herein byreference in their entireties.

SUMMARY OF THE DISCLOSURE

In some aspects, provided herein is a method comprising: contacting apopulation of pancreatic progenitor cells or precursors thereof with anepigenetic modifying compound, wherein the contacting results in apopulation of endocrine cells with an increased proportion ofchromogranin A-positive (CHGA+) cells or an increased proportion ofC-peptide-positive and NKX6.1-positive (C-PEP+, NKX6.1+) cells ascompared to a corresponding population of endocrine cells which is notcontacted with the epigenetic modifying compound.

In some aspects, provided herein is a method comprising: contacting apopulation of pancreatic progenitor cells or precursors thereof with anepigenetic modifying compound, wherein the contacting results in apopulation of endocrine cells with a reduced proportion of cellsexpressing VMAT or Cdx2 as compared to a corresponding population ofendocrine cells which is not contacted with the epigenetic modifyingcompound.

In some cases, the epigenetic modifying compound comprises one or moreof a DNA methylation inhibitor, a histone acetyltransferase inhibitor, ahistone deacetylase inhibitor, a histone methyltransferase inhibitor, ora bromodomain inhibitor. In some cases, the epigenetic modifyingcompound comprises a histone methyltransferase inhibitor. In some cases,the histone methyltransferase inhibitor is an EZH2 inhibitor. In somecases, the histone methyltransferase inhibitor is selected from thegroup consisting of DZNep, GSK126, and EPZ6438. In some cases, thehistone methyltransferase inhibitor is DZNep. In some cases, aconcentration of the DZNep that is contacted to the population ofpancreatic progenitor cells or precursors thereof is from about 0.05 μMto about 50 μM, about 0.1 μM to about 10 μM, about 0.5 μM to about 5 μM,about 0.75 μM to about 2.5 μM, or about 1 μM to about 2 μM. In somecases, the concentration of the DZNep is at least about 0.5 μM. In somecases, the concentration of the DZNep is about 1 μM. In some cases, theepigenetic modifying compound comprises a histone deacetylase (HDAC)inhibitor. In some cases, the HDAC inhibitor is a Class I HDACinhibitor, a Class II HDAC inhibitor, or a combination thereof. In somecases, the HDAC inhibitor is selected from the group consisting ofKD5170, MC1568, and TMP195. In some cases, the HDAC inhibitor is KD5170.In some cases, the epigenetic modifying compound comprises an HDACinhibitor and an EZH2 inhibitor. In some cases, the epigenetic modifyingcompound comprises DZNep and KD5170. In some cases, the method isperformed in vitro.

In some cases, the method further comprises contacting the population ofpancreatic progenitor cells or precursors thereof with an agent selectedfrom the group consisting of (i) a SHH pathway inhibitor, (ii) aretinoic acid (RA) signaling pathway activator, (iii) a γ-secretaseinhibitor, (iv) a growth factor from the epidermal growth factor (EGF)family, (v) a bone morphogenetic protein (BMP) signaling pathwayinhibitor, (vi) a TGF-β signaling pathway inhibitor, (vii) a thyroidhormone signaling pathway activator, (viii) a protein kinase inhibitor,and (ix) a ROCK inhibitor. In some cases, (A) the SHH pathway inhibitorcomprises SANT1; (B) the RA signaling pathway activator comprisesretinoic acid; (C) the γ-secretase inhibitor comprises XXI; (D) thegrowth factor from the EGF family comprises betacellulin; (E) the BMPsignaling pathway inhibitor comprises LDN; (F) the TGF-β signalingpathway inhibitor comprises Alk5i II; (G) the thyroid hormone signalingpathway activator comprises GC-1; (H) the protein kinase inhibitorcomprises staurosporine; or (I) the ROCK inhibitor comprisesthiazovinin. In some cases, the method comprises contacting thepopulation of pancreatic progenitor cells or precursors thereof with anagent selected from the group consisting of betacellulin, thiazovinin,retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN and staurosporine. Insome cases, the contacting is for at least three days. In some cases,the contacting comprises contacting the population of pancreaticprogenitor cells or precursors thereof with the epigenetic modifyingcompound for a period of more than three days, and removing the SHHpathway inhibitor, the RA signaling pathway activator, or the growthfactor from the EGF family after the contacting with the population ofpancreatic progenitor cells or precursors thereof for first three daysof the period. In some cases, the contacting is for at least five days.In some cases, the contacting is for about seven days. In some cases, atleast one cell of the population of pancreatic progenitor cellsexpresses at least one of PDX1 and NKX6-1. In some cases, at least onecell of the population of pancreatic progenitor cells expresses bothPDX1 and NKX6-1. In some cases, at least one cell of the population ofendocrine cells expresses CHGA. In some cases, at least one cell of thepopulation of endocrine cells expresses C-peptide and NKX6.1. In somecases, the population of endocrine cells comprises a proportion of CHGA+cells that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 320%, 350%,380%, 400%, 420%, 450%, 480%, or 500% higher than a correspondingpopulation of endocrine cells which is not contacted with the epigeneticmodifying compound, as measured by flow cytometry. In some cases, thepopulation of endocrine cells comprises a proportion of C-PEP+, NKX6.1+cells that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 320%, 350%,380%, 400%, 420%, 450%, 480%, or 500% higher than a correspondingpopulation of endocrine cells which is not contacted with the epigeneticmodifying compound, as measured by flow cytometry. In some cases, thepopulation of endocrine cells comprises a proportion of cells expressingVMAT or Cdx2 that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 320%,350%, 380%, or 400% lower than a corresponding population of endocrinecells which is not contacted with the at least one epigenetic modifyingcompound, as measured by flow cytometry.

In some aspects, provided herein is a cell produced by any methodprovided herein.

In some aspects, provided herein is a composition comprising a cellpopulation, wherein the cell population comprises: (a) at least about20% cells expressing C-peptide and NKX6.1; (b) at least about 60% cellsexpressing CHGA; (c) at most about 20% cells expressing Cdx2; or (d) atmost about 45% cells expressing VMAT1, as measured by flow cytometry.

In some aspects, provided herein is a composition comprising a cellpopulation that comprises at least about 30% ISL1-positive,NKX6.1-positive cells and at most about 20% ISL1-negative,NKX6.1-negative cells, as measured by flow cytometry.

In some cases, the cell population comprises at least about 35%ISL1-positive, NKX6.1-positive cells. In some cases, the cell populationcomprises at least about 40% ISL1-positive, NKX6.1-positive cells. Insome cases, the cell population comprises at most about 15%ISL1-negative, NKX6.1-negative cells. In some cases, the compositioncomprises: (a) at least about 20% cells expressing C-peptide and NKX6.1;(b) at least about 60% cells expressing CHGA; and (c) at most about 20%cells expressing Cdx2, as measured by flow cytometry. In some cases, thecomposition comprises at most about 45% cells expressing VMAT1, asmeasured by flow cytometry. In some cases, the composition furthercomprises an epigenetic modifying compound. In some cases, theepigenetic modifying compound comprises one or more of a DNA methylationinhibitor, a histone acetyltransferase inhibitor, a histone deacetylaseinhibitor, a histone methyltransferase inhibitor, or a bromodomaininhibitor. In some cases, the epigenetic modifying compound comprises ahistone methyltransferase inhibitor. In some cases, the histonemethyltransferase inhibitor is an EZH2 inhibitor. In some cases, thehistone methyltransferase inhibitor is selected from the groupconsisting of DZNep, GSK126, and EPZ6438. In some cases, the histonemethyltransferase inhibitor is DZNep. In some cases, a concentration ofthe DZNep that is contacted to the population of pancreatic progenitorcells or precursors thereof is from about 0.05 μM to about 50 μM, about0.1 μM to about 10 μM, about 0.5 μM to about 5 μM, about 0.75 μM toabout 2.5 μM, or about 1 μM to about 2 μM. In some cases, theconcentration of the DZNep is at least about 0.5 μM. In some cases, theconcentration of the DZNep is about 1 μM. In some cases, the epigeneticmodifying compound comprises a histone deacetylase (HDAC) inhibitor. Insome cases, the HDAC inhibitor is a Class I HDAC inhibitor, a Class IIHDAC inhibitor, or a combination thereof. In some cases, the HDACinhibitor is selected from the group consisting of KD5170, MC1568, andTMP195. In some cases, the HDAC inhibitor is KD5170. In some cases, theepigenetic modifying compound comprises an HDAC inhibitor and an EZH2inhibitor. In some cases, the epigenetic modifying compound comprisesDZNep and KD5170. In some cases, the composition further comprises anagent selected from the group consisting of (i) a SHH pathway inhibitor,(ii) a retinoic acid (RA) signaling pathway activator, (iii) aγ-secretase inhibitor, (iv) a growth factor from the epidermal growthfactor (EGF) family, (v) a bone morphogenetic protein (BMP) signalingpathway inhibitor, (vi) a TGF-β signaling pathway inhibitor, (vii) athyroid hormone signaling pathway activator, (viii) a protein kinaseinhibitor, and (ix) a ROCK inhibitor. In some cases of the compositions,(A) the SHH pathway inhibitor comprises SANT1; (B) the RA signalingpathway activator comprises retinoic acid; (C) the γ-secretase inhibitorcomprises XXI; (D) the growth factor from the EGF family comprisesbetacellulin; (E) the BMP signaling pathway inhibitor comprises LDN; (F)the TGF-β signaling pathway inhibitor comprises Alk5i II; (G) thethyroid hormone signaling pathway activator comprises GC-1; (H) theprotein kinase inhibitor comprises staurosporine; or (I) the ROCKinhibitor comprises thiazovinin. In some cases, the compositioncomprises an agent selected from the group consisting of betacellulin,thiazovinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN andstaurosporine.

In some aspects, provided herein is a composition that comprises apancreatic progenitor cell, and at least one of a histone deacetylase(HDAC) inhibitor or a histone methyltransferase inhibitor.

In some cases, the composition further comprises an endocrine cell. Insome cases of the composition, the HDAC inhibitor is a Class I HDACinhibitor, a Class II HDAC inhibitor, or a combination thereof. In somecases, the HDAC inhibitor is selected from the group consisting ofKD5170, MC1568, and TMP195. In some cases, the HDAC inhibitor is KD5170.In some cases, a concentration of the KD5170 in the composition is fromabout 0.05 μM to about 50 μM, about 0.1 μM to about 10 μM, about 0.5 μMto about 5 μM, about 0.75 μM to about 2.5 μM, or about 1 μM to about 2μM. In some cases, the concentration of the KD5170 is at least 0.5 μM.In some cases, the concentration of the KD5170 is about 1 μM. In somecases, the histone methyltransferase inhibitor is an EZH2 inhibitor. Insome cases, the histone methyltransferase inhibitor is selected from thegroup consisting of DZNep, GSK126, and EPZ6438. In some cases, thehistone methyltransferase inhibitor is DZNep. In some cases, aconcentration of the DZNep in the composition is from about 0.05 μM toabout 50 μM, about 0.1 μM to about 10 μM, about 0.5 μM to about 5 μM,about 0.75 μM to about 2.5 μM, or about 1 μM to about 2 μM. In somecases, the concentration of the DZNep is at least 0.5 μM. In some cases,the concentration of the DZNep is about 1 μM. In some cases, the HDACinhibitor is KD5170 and the histone methyltransferase inhibitor isDZNep. In some cases, the composition is an in vitro composition. Insome cases, the composition further comprises an agent selected from thegroup consisting of (i) a SHH pathway inhibitor, (ii) a retinoic acid(RA) signaling pathway activator, (iii) a γ-secretase inhibitor, (iv) agrowth factor from the epidermal growth factor (EGF) family, (v) a bonemorphogenetic protein (BMP) signaling pathway inhibitor, (vi) a TGF-βsignaling pathway inhibitor, (vii) a thyroid hormone signaling pathwayactivator, (viii) a protein kinase inhibitor, and (ix) a ROCK inhibitor.In some cases, (A) the SHH pathway inhibitor comprises SANT1; (B) the RAsignaling pathway activator comprises retinoic acid; (C) the γ-secretaseinhibitor comprises XXI; (D) the growth factor from the EGF familycomprises betacellulin; (E) the BMP signaling pathway inhibitorcomprises LDN; (F) the TGF-β signaling pathway inhibitor comprises Alk5iII; (G) the thyroid hormone signaling pathway activator comprises GC-1;(H) the protein kinase inhibitor comprises staurosporine; or (I) theROCK inhibitor comprises thiazovinin. In some cases, the compositionfurther comprises an agent selected from the group consisting ofbetacellulin, thiazovinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1,LDN and staurosporine.

In some aspects, provided herein is a method comprising: contacting acell population comprising pancreatic progenitor cells or precursorsthereof with a histone methyltransferase inhibitor and generating a cellpopulation comprising endocrine cells; and maturing the cell populationcomprising endocrine cells to obtain at least one pancreatic β cell thatexhibits an in vitro glucose-stimulated insulin secretion response to aglucose challenge.

In some cases, the method comprises contacting the cell population withan agent selected from the group consisting of (i) a SHH pathwayinhibitor, (ii) a retinoic acid (RA) signaling pathway activator, (iii)a γ-secretase inhibitor, (iv) a growth factor from the epidermal growthfactor (EGF) family, (v) a bone morphogenetic protein (BMP) signalingpathway inhibitor, (vi) a TGF-β signaling pathway inhibitor, (vii) athyroid hormone signaling pathway activator, (viii) a protein kinaseinhibitor, and (ix) a ROCK inhibitor. In some cases, (A) the SHH pathwayinhibitor comprises SANT1; (B) the RA signaling pathway activatorcomprises retinoic acid; (C) the γ-secretase inhibitor comprises XXI;(D) the growth factor from the EGF family comprises betacellulin; (E)the BMP signaling pathway inhibitor comprises LDN; (F) the TGF-βsignaling pathway inhibitor comprises Alk5i II; (G) the thyroid hormonesignaling pathway activator comprises GC-1; (H) the protein kinaseinhibitor comprises staurosporine; or (I) the ROCK inhibitor comprisesthiazovinin. In some cases, the method comprises contacting the cellpopulation with an agent selected from the group consisting ofbetacellulin, thiazovinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1,LDN and staurosporine. In some cases, the method further comprisescontacting the cell population with a histone deacetylase (HDAC)inhibitor. In some cases, the HDAC inhibitor is KD5170. In some cases,the histone methyltransferase inhibitor is selected from the groupconsisting of DZNep, GSK126, and EPZ6438. In some cases, the histonemethyltransferase inhibitor is DZNep. In some cases, the contacting withthe histone methyltransferase inhibitor results in a populationcomprising the endocrine cells and having an increased proportion ofchromogranin A-positive (CHGA+) cells or an increased proportion ofC-peptide-positive and NKX6.1-positive (C-PEP+; NKX6.1+) cells ascompared to a corresponding population of endocrine cells which is notcontacted with the histone methyltransferase inhibitor. In some cases,the contacting with the histone methyltransferase inhibitor results in apopulation comprising the endocrine cells and having a reducedproportion of cells expressing VMAT or Cdx2 as compared to acorresponding population of endocrine cells which is not contacted withthe histone methyltransferase inhibitor. In some cases, the at least onecell of the pancreatic progenitor cells or precursors thereof expressesboth Pdx1 and NKX6.1. In some cases, the method further comprisesdifferentiating a plurality of stem cells in vitro to obtain the cellpopulation comprising the pancreatic progenitor cells or precursorsthereof.

In some aspects, provided herein is a pancreatic 13 cell generatedaccording to any method provided herein.

In some aspects, provided herein is a method, comprising: (a) contactinga population of Pdx1-negative, NKX6.1-negative primitive gut tube cellswith a bone morphogenetic protein (BMP) signaling pathway inhibitor anda growth factor from transformation growth factor β (TGF-β) superfamily,thereby generating a cell population that comprises Pdx1-positive,NKX6.1-positive pancreatic progenitor cells; and (b) contacting the cellpopulation comprising the Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells with an epigenetic modifying compound and generating acell population comprising endocrine cells.

In some cases, the BMP signaling pathway inhibitor comprises DMH-1, aderivative, analogue, or variant thereof. In some cases, a concentrationof the DMH-1 that is contacted to the population of Pdx1-negative,NKX6.1-negative primitive gut tube cells is about 0.01 μM to about 10μM, about 0.05 μM to about 5 μM, about 0.1 μM to about 1 μM, or about0.15 μM to about 0.5 μM. In some cases, a concentration of the DMH-1that is contacted to the population of Pdx1-negative, NKX6.1-negativeprimitive gut tube cells is about 0.25 μM DMH-1. In some cases, thegrowth factor from TGF-β superfamily comprises Activin A. In some cases,a concentration of the Activin A that is contacted to the population ofPdx1-negative, NKX6.1-negative primitive gut tube cells is about 0.5ng/mL to about 200 ng/mL, about 1 ng/mL to about 100 ng/mL, about 2ng/mL to about 50 ng/mL, or about 5 ng/mL to about 30 ng/mL. In somecases, a concentration of the Activin A that is contacted to thepopulation of Pdx1-negative, NKX6.1-negative primitive gut tube cells isat least about 5 ng/mL or at least about 10 ng/mL Activin A. In somecases, a concentration of the Activin A that is contacted to thepopulation of Pdx1-negative, NKX6.1-negative primitive gut tube cells isabout 20 ng/mL Activin A. In some cases, the contacting the populationof Pdx1-negative, NKX6.1-negative primitive gut tube cells furthercomprises contacting with an agent selected from the group consistingof: a growth factor from FGF family, a SHH pathway inhibitor, a RAsignaling pathway activator, a protein kinase C activator, and a ROCKinhibitor. In some cases, the epigenetic modifying compound comprises acompound selected from the group consisting of a DNA methylationinhibitor, a histone acetyltransferase inhibitor, a histone deacetylaseinhibitor, a histone methyltransferase inhibitor, and a bromodomaininhibitor. In some cases, the epigenetic modifying compound comprises ahistone methyltransferase inhibitor. In some cases, the histonemethyltransferase inhibitor is an EZH2 inhibitor. In some cases, thehistone methyltransferase inhibitor is selected from the groupconsisting of DZNep, GSK126, and EPZ6438. In some cases, the histonemethyltransferase inhibitor is DZNep. In some cases, a concentration ofthe DZNep that is contacted to the population of pancreatic progenitorcells or precursors thereof is from about 0.05 μM to about 50 μM, about0.1 μM to about 10 μM, about 0.5 μM to about 5 μM, about 0.75 μM toabout 2.5 μM, or about 1 μM to about 2 μM. In some cases, theconcentration of the DZNep is at least about 0.5 μM. In some cases, theconcentration of the DZNep is about 1 μM. In some cases, the epigeneticmodifying compound comprises a histone deacetylase (HDAC) inhibitor. Insome cases, the HDAC inhibitor is a Class I HDAC inhibitor, a Class IIHDAC inhibitor, or a combination thereof. In some cases, the HDACinhibitor is selected from the group consisting of KD5170, MC1568, andTMP195. In some cases, the HDAC inhibitor is KD5170. In some cases, themethod is performed in vitro. In some cases, the method furthercomprises contacting the population comprising the Pdx1-positive,NKX6.1-positive pancreatic progenitor cells with an agent selected fromthe group consisting of: (i) a SHH pathway inhibitor, (ii) a retinoicacid (RA) signaling pathway activator, (iii) a γ-secretase inhibitor,(iv) a growth factor from the epidermal growth factor (EGF) family, (v)a bone morphogenetic protein (BMP) signaling pathway inhibitor, (vi) aTGF-β signaling pathway inhibitor, (vii) a thyroid hormone signalingpathway activator, (viii) a protein kinase inhibitor, and (ix) a ROCKinhibitor. In some cases, (A) the SHH pathway inhibitor comprises SANT1;(B) the RA signaling pathway activator comprises retinoic acid; (C) theγ-secretase inhibitor comprises XXI; (D) the growth factor from the EGFfamily comprises betacellulin; (E) the BMP signaling pathway inhibitorcomprises LDN; (F) the TGF-β signaling pathway inhibitor comprises Alk5iII; (G) the thyroid hormone signaling pathway activator comprises GC-1;(H) the protein kinase inhibitor comprises staurosporine; or (I) theROCK inhibitor comprises thiazovinin. In some cases, the methodcomprises contacting the population comprising the Pdx1-positive,NKX6.1-positive pancreatic progenitor cells with an agent selected fromthe group consisting of: betacellulin, thiazovinin, retinoic acid,SANT1, XXI, Alk5i II, GC-1, LDN, and staurosporine. In some cases, thecontacting is for at least three days. In some cases, the contactingcomprises contacting the population comprising the Pdx1-positive,NKX6.1-positive pancreatic progenitor cells with the epigeneticmodifying compound for a period of more than three days, and removingthe SHH pathway inhibitor, the RA signaling pathway activator, or thegrowth factor from the EGF family after the contacting with thepopulation of pancreatic progenitor cells or precursors thereof forfirst three days of the period. In some cases, the contacting is for atleast five days. In some cases, the contacting is for about seven days.In some cases, the cell population that comprises Pdx1-positive,NKX6.1-positive pancreatic progenitor cells comprises at most about 10%cells expressing Cdx2, as measured by flow cytometry. In some cases, thecell population that comprises Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells comprises a smaller proportion of cells expressing Cdx2as compared to a corresponding cell population without contacting withthe bone morphogenetic protein (BMP) signaling pathway inhibitor and thegrowth factor from transformation growth factor 13 (TGF-β) superfamily.In some cases, the cell population comprising endocrine cells comprisesat least about 40% cells expressing ISL1 and NKX6.1, as measured by flowcytometry. In some cases, the cell population comprising endocrine cellscomprises a higher proportion of cells expressing ISL1 and NKX6.1 ascompared to a corresponding cell population without the contacting withthe bone morphogenetic protein (BMP) signaling pathway inhibitor and thegrowth factor from transformation growth factor β (TGF-β) superfamily.In some cases, the cell population comprising endocrine cells comprisesat most about 15% ISL1-negative, NKX6.1-negative cells, as measured byflow cytometry. In some cases, the cell population comprising endocrinecells comprises a smaller proportion of ISL1-negative, NKX6.1-negativecells as compared to a corresponding cell population without thecontacting with the bone morphogenetic protein (BMP) signaling pathwayinhibitor and the growth factor from transformation growth factor 13(TGF-β) superfamily. In some cases, the method further comprisescryopreserving the cell population comprising endocrine cells. In somecases, the cell population comprising endocrine cells is a cell cluster,and the method further comprises: (a) dissociating a plurality of cellsfrom the cell cluster; and (b) culturing the plurality of cells from (a)in a reaggregation culture medium and allowing at least a portion of theplurality of cells to form a second cell cluster. In some cases, thedissociating does not comprise subjecting the plurality of cells to flowcytometry. In some cases, the reaggregation culture medium isserum-free. In some cases, the reaggregation culture medium does notcomprise exogenous differentiation factors. In some cases, the methodfurther comprises maturing the endocrine cells in vitro to obtain atleast one pancreatic β cell that exhibits an in vitro glucose-stimulatedinsulin secretion response to a glucose challenge. In some cases, thematuring is performed in a serum-free medium. In some cases, thematuring is performed in a xeno-free medium. In some cases, the maturingis performed in a culture medium that does not comprise exogenousdifferentiation factors. In some cases, the maturing is performed in apresence of human serum albumin (HSA). In some cases, the HSA is presentat a concentration of about 0.1% to about 5%, about 0.5% to about 2%. Insome cases, the HSA is present at a concentration of about 1%.

In some cases, the methods provided herein comprise A method,comprising: (a) differentiating pluripotent stem cells in a populationinto definitive endoderm cells by contacting the pluripotent stem cellswith a growth factor from TGF-β superfamily and a WNT signaling pathwayactivator; (b) differentiating at least some of the definitive endodermcells into primitive gut tube cells by contacting the definitiveendoderm cells with a growth factor from FGF family; (c) differentiatingat least some of the primitive gut tube cells into Pdx1-positivepancreatic progenitor cells by contacting the primitive gut tube cellswith a ROCK inhibitor, a growth factor from FGF family, a BMP signalingpathway inhibitor, a PKC activator, a retinoic acid signaling pathwayactivator, a SHH pathway inhibitor, and a growth factor from TGF-βsuperfamily; (d) differentiating at least some of the Pdx1-positivepancreatic progenitor cells into Pdx1-positive, NKX6.1-positivepancreatic progenitor cells by contacting the Pdx1-positive pancreaticprogenitor cells with a ROCK inhibitor, a growth factor from TGFβsuperfamily, a growth factor from FGF family, a RA signaling pathwayactivator, and a SHH pathway inhibitor; and (e) differentiating at leastsome of the Pdx1-positive, NKX6.1-positive pancreatic progenitor cellsinto a cell population comprising at least one NKX6.1+ and C-peptide+cell by contacting the Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells with a TGF-β signaling pathway inhibitor, a growthfactor from EGF family, a RA signaling pathway activator, a SHH pathwayinhibitor, a TH signaling pathway activator, a γ-secretase inhibitor, aprotein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathwayinhibitor, and an epigenetic modifying compound.

In some aspects, provided herein is a cell population comprisingendocrine cells generated according to any method provided herein. Insome aspects, provided herein is a cell population comprising SC-β cellsthat are generated according to any method provided herein.

In some aspects, provided herein is a method, comprising: exposing an invitro cell population comprising endocrine cells to irradiation at adose of about 100 rads to about 100,000 rads for a time period of about1 min to about 60 min.

In some aspects, provided herein is a method of reducing cellproliferation, comprising exposing to irradiation a cell populationcomprising stem cells, definitive endoderm cells, primitive gut tubecells, pancreatic progenitor cells, or endocrine cells, wherein theirradiation results in a cell population that has reduced proliferativecapability as compared to a corresponding cell population that is notsubject to irradiation.

In some cases, the cell population is exposed to irradiation at about100 rads to about 50,000 rads, about 100 rads to about 25,000 rads,about 100 rads to about 10,000 rads, about 250 rads to about 25,000rads, about 500 rads to about 25,000 rads, about 1,000 rads to about25,000 rads, about 2,500 rads to about 25,000 rads, about 5,000 rads toabout 25,000 rads, or about 10,000 rads to about 15,000 rads. In somecases, the cell population is exposed to irradiation at about 10,000rads. In some cases, the cell population is exposed to irradiation forabout 1 min to about 55 min, about 1 min to about 50 min, about 1 min toabout 45 min, about 1 min to about 40 min, about 1 min to about 35 min,about 1 min to about 30 min, about 1 min to about 25 min, about 1 min toabout 20 min, about 1 min to about 10 min, about 1 min to about 5 min,about 10 min to about 55 min, about 15 min to about 55 min, about 20 minto about 55 min, about 25 min to about 55 min, about 30 min to about 55min, about 20 min to about 40 min, or about 25 min to about 35 min. Insome cases, the cell population is exposed to irradiation for about 30min. In some cases, the irradiation comprises ionizing irradiation. Insome cases, the ionizing irradiation comprises gamma ray, x-ray,ultraviolet radiation, alpha ray, beta ray, or neutron ray. In somecases, the irradiation results in a cell population with reducedproliferation as compared to a corresponding cell cluster that is notsubject to the irradiation. In some cases, the cell population comprisesa second cell cluster having a diameter of about 50 μm to about 500 μm,about 50 μm to about 300 μm, about 50 μm to about 200 μm, about 50 μm toabout 150 μm, about 600 μm to about 150 μm, about 700 μm to about 150μm, about 80 μm to about 150 μm, or about 60 μm to about 100 μm. In somecases, the method further comprises (a) dissociating a plurality ofcells from a first cell cluster; and (b) culturing the plurality ofcells from (a) in a reaggregation culture medium and allowing at least aportion of the plurality of cells to form the second cell cluster. Insome cases, the dissociating does not comprise subjecting the pluralityof cells to flow cytometry. In some cases, the first cell cluster isobtained by dissociating a third cell cluster and culturing cellsdissociated from the third cell cluster to form the first cell cluster.In some cases, the cell cluster is cryopreserved prior to theirradiation, wherein the method further comprises thawing thecryopreserved cell cluster prior to the irradiation. In some cases, thecell cluster is cryopreserved while being subject to the irradiation. Insome cases, method further comprises thawing the cryopreserved cellcluster after irradiation and differentiating at least some of theendocrine cells. In some cases, the method further comprises obtainingthe cell population comprising the endocrine cells by differentiatingpancreatic progenitor cells or precursor thereof in vitro. In somecases, the method further comprises differentiating stem cells in vitro,thereby generating the cell population comprising the endocrine cells.In some cases, the method further comprises maturing at least some ofthe endocrine cells in vitro into pancreatic β cells, thereby generatinga cell population comp. In some cases, the method further comprisesimplanting the pancreatic β cells into a subject in need thereof. Insome cases, the implanted pancreatic β cells are configured to controlblood glucose level in the subject for at least about 50 days, 60 days,70 days, 80 days, 90 days, or longer.

In some aspects, provided herein is a cell population comprisingendocrine cells generated according to any method of irradiation asdescribed herein. In some aspects, provided herein is a cell populationcomprising pancreatic β cells generated according to any method ofirradiation as described herein.

In some aspects, provided herein is a method comprising: contacting apopulation of pancreatic progenitor cells or precursors thereof with acomposition comprising at least one epigenetic modifying compound,wherein the contacting results in a population of endocrine cells with areduced proportion of cells expressing VMAT or Cdx2 as compared to acorresponding population of endocrine cells which is not contacted withthe at least one epigenetic modifying compound.

In some embodiments, the at least one epigenetic modifying compoundcomprises one or more of a DNA methylation inhibitor, a histoneacetyltransferase inhibitor, a histone deacetylase inhibitor, a histonemethyltransferase inhibitor, or a bromodomain inhibitor. In someembodiments, the at least one epigenetic modifying compound comprises ahistone methyltransferase inhibitor. In some embodiments, the histonemethyltransferase inhibitor is an EZH2 inhibitor.

In some embodiments, the histone methyltransferase inhibitor is at leastone of DZNep, GSK126, or EPZ6438. In some embodiments, the histonemethyltransferase inhibitor is DZNep. In some embodiments, aconcentration of the DZNep in the composition is greater than 0.1 μM. Insome embodiments, the concentration of the DZNep is at least 0.5 μM. Insome embodiments, the concentration of the DZNep is about 1 μM. In someembodiments, the at least one epigenetic modifying compound comprises ahistone deacetylase (HDAC) inhibitor. In some embodiments, the HDACinhibitor is a Class I HDAC inhibitor, a Class II HDAC inhibitor, or acombination thereof. In some embodiments, the HDAC inhibitor is at leastone of KD5170, MC1568, or TMP195. In some embodiments, the HDACinhibitor is KD5170. In some embodiments, the at least one epigeneticmodifying compound comprises an HDAC inhibitor and an EZH2 inhibitor. Insome embodiments, the at least one epigenetic modifying compoundcomprises DZNep and KD5170.

In some embodiments, at least one of the cells expressing VMAT is INS⁻in the method provided herein. In some embodiments, at least some cellsof the population of pancreatic progenitor cells differentiate into apopulation of PH cells. In some embodiments, an increased proportion ofcells of the population of endocrine cells are NKX6.1⁻ or ChromA⁺ ascompared to the corresponding population of endocrine cells which is notcontacted with the at least one epigenetic modifying compound. In someembodiments, at least one cell of the increased proportion of cells isNKX6.1⁻ and ChromA⁺. In some embodiments, at least some cells of thepopulation of pancreatic progenitor cells differentiate into apopulation of β cells. In some embodiments, the β cells are stem-cellderived β (SC-β) cells. In some embodiments, the β cells express C-PEPand NKX6-1. In some embodiments, the β cells exhibit an in vitroglucose-stimulated insulin secretion response to a glucose challenge.

In some embodiments, the composition of the method described hereincomprises at least one of betacellulin, thiazovinin, retinoic acid,SANT1, XXI, Alk5i II, GC-1, LDN or staurosporine. In some embodiments,the contacting is for at least three days. In some embodiments, thecontacting is for at least five days. In some embodiments, thecontacting is for about seven days.

In some embodiments, at least one pancreatic progenitor cell of thepopulation of pancreatic progenitor cells expresses at least one of PDX1and NKX6-1. In some embodiments, at least one endocrine cell of thepopulation of endocrine cells expresses CHGA.

Provided herein is an endocrine cell produced by any of the methoddescribed herein.

Provided herein is a composition that comprises a pancreatic progenitorcell, a histone deacetylase (HDAC) inhibitor, a histonemethyltransferase inhibitor and optionally an endocrine cell. In someembodiments, the HDAC inhibitor is a Class I HDAC inhibitor, a Class IIHDAC inhibitor, or a combination thereof. In some embodiments, the HDACinhibitor is at least one of KD5170, MC1568, or TMP195. In someembodiments, the HDAC inhibitor is KD5170. In some embodiments, aconcentration of the KD5170 in the composition is at least 0.1 μM. Insome embodiments, the concentration of the KD5170 is at least 0.5 μM. Insome embodiments, the concentration of the KD5170 is about 1 μM. In someembodiments, the histone methyltransferase inhibitor is an EZH2inhibitor. In some embodiments, the histone methyltransferase inhibitoris at least one of DZNep, GSK126, or EPZ6438. In some embodiments, thehistone methyltransferase inhibitor is DZNep. In some embodiments, aconcentration of the DZNep is at least 0.1 μM. In some embodiments, theconcentration of the DZNep is at least 0.5 μM. In some embodiments, theconcentration of the DZNep is about 1 μM. In some embodiments, the HDACinhibitor is KD5170 and the histone methyltransferase inhibitor isDZNep. In some embodiments, the composition is an in vitro composition.In some embodiments, the composition further comprises at least one ofbetacellulin, thiazovinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1,LDN or staurosporine.

Provided herein is a method comprising contacting a pancreaticprogenitor cell or precursor thereof with a histone deacetylase (HDAC)inhibitor and a histone methyltransferase inhibitor, wherein thecontacting induces differentiation of the pancreatic progenitor cell. Insome embodiments, the pancreatic progenitor cell differentiates into a βcell. In some embodiments, the β cell is a stem-cell derived β (SC-β)cell. In some embodiments, C-PEP and NKX6-1. In some embodiments, the βcells exhibit an in vitro glucose-stimulated insulin secretion responseto a glucose challenge. In some embodiments, the HDAC inhibitor is aClass I HDAC inhibitor, a Class II HDAC inhibitor, or a combinationthereof. In some embodiments, the HDAC inhibitor is at least one ofKD5170, MC1568, or TMP195. In some embodiments, the HDAC inhibitor isKD5170. In some embodiments, the histone methyltransferase inhibitor isan EZH2 inhibitor. In some embodiments, the histone methyltransferaseinhibitor is at least one of DZNep, GSK126, or EPZ6438. In someembodiments, the histone methyltransferase inhibitor is DZNep. In someembodiments, the HDAC inhibitor is KD5170 and the histonemethyltransferase inhibitor is DZNep. In some embodiments, the method isperformed in vitro.

Provided herein is a method comprising contacting a pancreaticprogenitor cell or precursor thereof with KD5170 in an amount sufficientto result in differentiation of the cell. In some embodiments, themethod further comprises contracting the pancreatic progenitor cell witha histone methyltransferase inhibitor. In some embodiments, the histonemethyltransferase inhibitor is at least one of DZNep, GSK126, orEPZ6438. In some embodiments, the histone methyltransferase inhibitor isDZNep. In some embodiments, the pancreatic progenitor celldifferentiates into an endocrine cell. In some embodiments, thepancreatic progenitor cell differentiates into a β cell. In someembodiments, the β cell is a stem-cell derived β (SC-β) cell. In someembodiments, the β cell expresses C-PEP and NKX6-1. In some embodiments,the β cell exhibits an in vitro glucose-stimulated insulin secretionresponse to a glucose challenge.

Provided herein is a method comprising: (a) differentiating a pluralityof stem cells in vitro to obtain a cell population comprising pancreaticprogenitor cells or precursors thereof; (b) contacting in vitro the cellpopulation with a histone deacetylase (HDAC) inhibitor to generate atleast one endocrine cell; and (c) maturing the endocrine cell in vitroto obtain at least one SC-β cell. In some embodiments, the stem cellsare human pluripotent stem cells. In some embodiments, the methodfurther comprises contacting the cell population with at least one ofbetacellulin, thiazovinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1,LDN or staurosporine. In some embodiments, the SC-β cell expresses C-PEPand NKX6-1. In some embodiments, the SC-β cell exhibits an in vitroglucose-stimulated insulin secretion response to a glucose challenge. Insome embodiments, the method further comprises contracting the cellpopulation with a histone methyltransferase inhibitor. In someembodiments, the histone methyltransferase inhibitor is at least one ofDZNep, GSK126, or EPZ6438. In some embodiments, the histonemethyltransferase inhibitor is DZNep. In some embodiments, the HDACinhibitor is KD5170.

Provided herein is a method comprising contacting a cell populationcomprising pancreatic progenitor cells or precursors thereof with ahistone methyltransferase inhibitor in vitro in an amount sufficient togenerate endocrine cells; and maturing the endocrine cells in vitro toobtain at least one SC-β cell that exhibits an in vitroglucose-stimulated insulin secretion response to a glucose challenge. Insome embodiments, the method further comprises differentiating aplurality of stem cells in vitro to obtain the cell populationcomprising the pancreatic progenitor cells or precursors thereof. Insome embodiments, the method further comprises contacting the cellpopulation with at least one of betacellulin, thiazovinin, retinoicacid, SANT1, XXI, Alk5i II, GC-1, LDN or staurosporine. In someembodiments, the method further comprises contacting the cell populationwith a histone deacetylase (HDAC) inhibitor. In some embodiments, theHDAC inhibitor is KD5170. In some embodiments, the histonemethyltransferase inhibitor is at least one of DZNep, GSK126, orEPZ6438. In some embodiments, the histone methyltransferase inhibitor isDZNep.

Provided herein is a method for selecting a target cell from apopulation of cells comprising (i) contacting the target cell with astimulating compound, wherein the contacting induces a selectable markerof the target cell to localize to a cell surface of the target cell; and(ii) selecting the target cell based on the localization of theselectable marker at the cell surface. In some embodiments, theselectable marker comprises PSA-NCAM. In some embodiments, the selectingthe target cell is by cell sorting. In some embodiments, the selectingcomprises contacting the selectable marker of the target cell with anantigen binding polypeptide when the selectable marker is localized tothe surface of the target cell. In some embodiments, the antigen bindingpolypeptide comprises an antibody. In some embodiments, the antigenbinding polypeptide binds to the PSA-NCAM. In some embodiments, themethod further comprises treating the population of cells with acompound that removes the selectable marker from a cell surface of atleast one cell of the population of cells. In some embodiments, thepopulation of cells is treated with the compound prior to the contactingthe target cell with the stimulating compound. In some embodiments, thecompound cleaves the selectable marker from the cell surface of the atleast one cell. In some embodiments, the compound is an enzyme. In someembodiments, the compound is an endosialidase. In some embodiments, theendosialidase is endoneuraminidase (Endo-N). In some embodiments, thetarget cell is an endocrine cell. In some embodiments, the stimulatingcompound comprises at least one of arginine or glucose. In someembodiments, the endocrine cell is a β cell. In some embodiments, the βcell is an SC-β cell. In some embodiments, the stimulating compoundcomprises isoproterenol. In some embodiments, the endocrine cell is anEC cell. In some embodiments, one or more cells of the population ofcells fails to localize the selectable marker to a cell surface whencontacted with the stimulating compound. In some embodiments, thestimulating compound is at least one of glucose or arginine and the oneor more cells is an EC cell. In some embodiments, the stimulatingcompound is isoproterenol and the one or more cells is a β cell. In someembodiments, the selecting the target cell separates the target cellfrom the one or more cells of the population of cells.

Provided herein is a method comprising: contacting a population ofpancreatic progenitor cells or precursors thereof with a compositioncomprising at least one epigenetic modifying compound, wherein thecontacting results in an increased proportion of islet cells as comparedto a corresponding population of pancreatic progenitor cells which isnot contacted with the at least one epigenetic modifying compound. Insome embodiments, the islet cells comprise at least one β cell. In someembodiments, the β cell comprises an SC-β cell. In some embodiments, theSC-β cell exhibits an in vitro glucose-stimulated insulin secretionresponse to a glucose challenge. In some embodiments, the islet cellscomprise at least one alpha cell. In some embodiments, the islet cellscomprise a delta cell. In some embodiments, the islet cells comprise apolyhormonal (PH) cell. In some embodiments, the method furthercomprises differentiating a plurality of stem cells in vitro to obtainthe population of pancreatic progenitor cells or precursors thereof. Insome embodiments, the stem cells are human pluripotent stem cells. Insome embodiments, the at least one epigenetic modifying compoundcomprises one or more of a DNA methylation inhibitor, a histoneacetyltransferase inhibitor, a histone deacetylase inhibitor, a histonemethyltransferase inhibitor, or a bromodomain inhibitor. In someembodiments, the at least one epigenetic modifying compound comprises ahistone methyltransferase inhibitor. In some embodiments, the histonemethyltransferase inhibitor is an EZH2 inhibitor. In some embodiments,the histone methyltransferase inhibitor is at least one of DZNep,GSK126, or EPZ6438. In some embodiments, the histone methyltransferaseinhibitor is DZNep. In some embodiments, a concentration of the DZNep inthe composition is greater than 0.1 μM. In some embodiments, theconcentration of the DZNep is at least 0.5 μM. In some embodiments, theconcentration of the DZNep is about 1 μM. In some embodiments, the atleast one epigenetic modifying compound comprises a histone deacetylase(HDAC) inhibitor. In some embodiments, the HDAC inhibitor is a Class IHDAC inhibitor, a Class II HDAC inhibitor, or a combination thereof. Insome embodiments, the HDAC inhibitor is at least one of KD5170, MC1568,or TMP195. In some embodiments, the HDAC inhibitor is KD5170. In someembodiments, the at least one epigenetic modifying compound comprises anHDAC inhibitor and an EZH2 inhibitor. In some embodiments, the at leastone epigenetic modifying compound comprises DZNep and KD5170.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the present disclosure are set forth with particularityin the appended claims. A better understanding of the features andadvantages of the present will be obtained by reference to the followingdetailed description that sets forth illustrative embodiments, in whichthe principles of the disclosure are utilized, and the accompanyingdrawings of which:

FIG. 1 shows chemical structures of 3-deazaneplanocin A hydrochloride(DZNep), GSK126, EPZ6438.

FIG. 2 shows chemical structures of KD5170 and MC1568.

FIG. 3 shows chemical structures of methyltransferase inhibitors.

FIG. 4 shows a chemical structure of DZNep analogues.

FIG. 5 is a summary of differentiation stages as described herein.

FIG. 6 is a schematic of directed differentiation from hPSC into INS+cells, comprising inhibition of histone methylation and deacetylation ofstage 5 as described herein. DE: definitive endoderm; PGT; primitive guttube; PPT1; early pancreatic progenitor; PPT2; PDX1+/NKX6.1+ pancreaticprogenitors; EN: NKX6.1/C peptide+ cells; SC-β: stem cell derived betacell.

FIG. 7 shows that stage 5 cells express NGN3 to initiate stem cell (SC)islet cell differentiation.

FIG. 8 shows that inhibition of EZH2 or HDAC in stage 5 increasesendocrine cells and SC-β cell.

FIG. 9 shows that combined inhibition of EZH2 and HDAC in stage 5significantly increases endocrine cells.

FIG. 10 shows that combined inhibition of EZH2 and HDAC in stage 5significantly increases endocrine cells.

FIG. 11 shows that combined inhibition of EZH2 and HDAC in stage 5significantly increases SC β cells.

FIG. 12 shows increased endocrine cells in stage 5 (n=2).

FIG. 13 shows combined EZH2 and HDAC inhibition increases neurogenin3+progenitors in stage 5.

FIG. 14 shows that DZNep outperforms other EZH2 inhibitors.

FIG. 15 shows that KD5170 outperforms other HDAC inhibitors.

FIG. 16 shows that combined inhibition of EZH2 and HDAC in stage 5increases NKX6-1+ progenitors.

FIG. 17 is an experiment outline for testing EZH2 and HDAC inhibitors instage 5.

FIG. 18 is a schematic of candidate screen set up.

FIG. 19 shows specific decrease in (VMAT1+ INS−) EC population.

FIG. 20 shows concomitant increase of (NKX6.1− ChromA+) PH cells. PH:polyhormonal cells.

FIG. 21 shows that the (NKX6.1+ INS+)SC-β population percentage is notaffected.

FIG. 22 shows different plates of cell surface marker discovery screen.

FIG. 23 shows MACS marker screening of stage 5.

FIG. 24 shows a schematic of SC-beta cell labeling and MACS makerscreening.

FIG. 25 shows that PSA-NCAM microbead based soring enriches on-targetcells and reduces SOX9+ cells.

FIG. 26 shows that EC cells (VMAT1+) remain after PSA-NCAM sorting.

FIG. 27 shows that PSA-NCAM expression decreases significantly uponEndo-N enzyme treatment. Endo-N is an endosialidase which degradesrapidly and specifically linear polymers of sialic acid withα-2,8-linkage with a minimum length of 7-9 residues characteristic ofsialic acid residues associated with NCAM. Cleavage of PSA on NCAM inphysiological conditions.

FIG. 28 is a schematic of removing EC cells using PSA-NCAM microbeadssorting.

FIG. 29 shows that EC cells can arise from intestinal progenitorspecified early in the differentiation process.

FIG. 30 shows that low OCT4 percentage at stage 0 complete leads tohigher CDX2 percentage in later stages. High Oct4% is required forrobust differentiation. Variability in Sox17 induction remains even withhigh Oct4%.

FIG. 31 is a schematic of compound screening based approach to identifyinhibitors of EC cell differentiation.

FIG. 32 is a schematic of compounds screening on stage 5 cells.

FIG. 33 shows irradiation dose dependent reduction of proliferatingcells.

FIG. 34 shows that high dose gamma irradiation had no significant impacton SC-islet composition and function.

FIG. 35 shows that cryopreserved SC-islets lost ability to control BGafter 60 days after irradiation.

FIG. 36 shows beta cell numbers in irradiated sample as compared tocontrol sample.

FIG. 37 shows glycemic control maintained by implanted irradiated mRAislet cells.

FIG. 38 shows glycemic control in all animals with irradiated SC-islets.

FIG. 39 shows diagrams of two exemplary protocols (v11 and v12) fordifferentiating human pluripotent stem cells into stem cell-derivedpancreatic β cells according to the present disclosure.

FIG. 40 shows that v12 protocol generated a higher proportion of cellsexpressing ISL1 and NKX6.1 (ISL1-positive, NKX6.1-positive) and a lowerproportion of ISL-negative, NKX6.1-negative cells at Stage 5, and alower proportion of CDX2-positive cells at Stage 4, as compared to v11protocol.

FIG. 41 shows that cell clusters generated via v12 protocol had a higherrecovery yield after cryopreservation as compared to v11 protocol.

FIG. 42 shows that v11 and v12 protocols generated comparable proportionof SC-β cells.

FIG. 43 shows that v11 and v12 protocols generated cell clusters havingcomparable GSIS response and insulin content.

FIG. 44 summarizes insulin release performance of an exemplary cellpopulation generated according to the methods provided herein in abioreactor in response to low glucose (LG), high glucose (HG), andpotassium chloride (KCl) challenges, respectively, and the insulincontent of the cell populations.

DETAILED DESCRIPTION OF THE DISCLOSURE

The following description and examples illustrate embodiments of thepresent disclosure in detail. It is to be understood that thisdisclosure is not limited to the particular embodiments described hereinand as such can vary. Those of skill in the art will recognize thatthere are numerous variations and modifications of this disclosure,which are encompassed within its scope.

All terms are intended to be understood as they would be understood by aperson skilled in the art. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which the disclosurepertains.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Although various features of the present disclosure can be described inthe context of a single embodiment, the features can also be providedseparately or in any suitable combination. Conversely, although thepresent disclosure can be described herein in the context of separateembodiments for clarity, the present disclosure can also be implementedin a single embodiment.

The following definitions supplement those in the art and are directedto the current application and are not to be imputed to any related orunrelated case, e.g., to any commonly owned patent or application.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice for testing of the presentdisclosure, the preferred materials and methods are described herein.Accordingly, the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Definitions

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. It must be noted that, as used in thespecification, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

In this application, the use of “or” means “and/or” unless statedotherwise. The terms “and/or” and “any combination thereof” and theirgrammatical equivalents as used herein, can be used interchangeably.These terms can convey that any combination is specificallycontemplated. Solely for illustrative purposes, the following phrases“A, B, and/or C” or “A, B, C, or any combination thereof” can mean “Aindividually; B individually; C individually; A and B; B and C; A and C;and A, B, and C.” The term “or” can be used conjunctively ordisjunctively, unless the context specifically refers to a disjunctiveuse.

Furthermore, use of the term “including” as well as other forms, such as“include”, “includes,” and “included,” is not limiting.

Reference in the specification to “some embodiments,” “an embodiment,”“one embodiment” or “other embodiments” means that a particular feature,structure, or characteristic described in connection with theembodiments is included in at least some embodiments, but notnecessarily all embodiments, of the present disclosures.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps. It is contemplated that any embodimentdiscussed in this specification can be implemented with respect to anymethod or composition of the present disclosure, and vice versa.Furthermore, compositions of the present disclosure can be used toachieve methods of the present disclosure.

The term “about” in relation to a reference numerical value and itsgrammatical equivalents as used herein can include the numerical valueitself and a range of values plus or minus 10% from that numericalvalue.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, up to 10%, up to 5%, or up to 1% of a given value. In anotherexample, the amount “about 10” includes 10 and any amounts from 9 to 11.In yet another example, the term “about” in relation to a referencenumerical value can also include a range of values plus or minus 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Alternatively,particularly with respect to biological systems or processes, the term“about” can mean within an order of magnitude, preferably within 5-fold,and more preferably within 2-fold, of a value. Where particular valuesare described in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

The term “diabetes” and its grammatical equivalents as used herein canrefer to is a disease characterized by high blood sugar levels over aprolonged period. For example, the term “diabetes” and its grammaticalequivalents as used herein can refer to all or any type of diabetes,including, but not limited to, type 1, type 2, cystic fibrosis-related,surgical, gestational diabetes, and mitochondrial diabetes. In somecases, diabetes can be a form of hereditary diabetes.

The term “endocrine cell(s),” if not particularly specified, can referto hormone-producing cells present in the pancreas of an organism, suchas “islet”, “islet cells”, “islet equivalent”, “islet-like cells”,“pancreatic islets” and their grammatical equivalents. In an embodiment,the endocrine cells can be differentiated from pancreatic progenitorcells or precursors. Islet cells can comprise different types of cells,including, but not limited to, pancreatic α cells, pancreatic β cells,pancreatic δ cells, pancreatic F cells, and/or pancreatic cells. Isletcells can also refer to a group of cells, cell clusters, or the like.

The terms “progenitor” and “precursor” cell are used interchangeablyherein and refer to cells that have a cellular phenotype that is moreprimitive (e.g., is at an earlier step along a developmental pathway orprogression than is a fully differentiated cell) relative to a cellwhich it can give rise to by differentiation. Often, progenitor cellscan also have significant or very high proliferative potential.Progenitor cells can give rise to multiple distinct differentiated celltypes or to a single differentiated cell type, depending on thedevelopmental pathway and on the environment in which the cells developand differentiate.

A “precursor thereof” as the term related to an insulin-positiveendocrine cell can refer to any cell that is capable of differentiatinginto an insulin-positive endocrine cell, including for example, apluripotent stem cell, a definitive endoderm cell, a primitive gut tubecell, a pancreatic progenitor cell, or endocrine progenitor cell, whencultured under conditions suitable for differentiating the precursorcell into the insulin-positive endocrine cell.

The term “exocrine cell” as used herein can refer to a cell of anexocrine gland, i.e. a gland that discharges its secretion via a duct.In particular embodiments, an exocrine cell can refer to a pancreaticexocrine cell, which is a pancreatic cell that can produce enzymes thatare secreted into the small intestine. These enzymes can help digestfood as it passes through the gastrointestinal tract. Pancreaticexocrine cells are also known as islets of Langerhans, which can secretetwo hormones, insulin and glucagon. A pancreatic exocrine cell can beone of several cell types; α-2 cells (which can produce the hormoneglucagon); or β cells (which can manufacture the hormone insulin); andα-1 cells (which can produce the regulatory agent somatostatin).Non-insulin-producing exocrine cells, as the term is used herein, canrefer to α-2 cells or α-1 cells. The term pancreatic exocrine cellsencompasses “pancreatic endocrine cells” which can refer to a pancreaticcell that produces hormones (e.g., insulin (produced from β cells),glucagon (produced by alpha-2 cells), somatostatin (produced by deltacells) and pancreatic polypeptide (produced by F cells) that aresecreted into the bloodstream.

The terms “stem cell-derived β cell,” “SC-β cell,” “functional β cell,”“functional pancreatic β cell,” “mature SC-β cell,” and theirgrammatical equivalents can refer to cells (e.g., non-native pancreaticβ cells) that display at least one marker indicative of a pancreatic βcell (e.g., PDX-1 or NKX6.1), expresses insulin, and display a glucosestimulated insulin secretion (GSIS) response characteristic of anendogenous mature β cell. In some embodiments, the terms “SC-β cell” and“non-native β cell” as used herein are interchangeable. In someembodiments, the “SC-β cell” comprises a mature pancreatic cell. It isto be understood that the SC-β cells need not be derived (e.g.,directly) from stem cells, as the methods of the disclosure are capableof deriving SC-β cells from any insulin-positive endocrine cell orprecursor thereof using any cell as a starting point (e.g., one can useembryonic stem cells, induced-pluripotent stem cells, progenitor cells,partially reprogrammed somatic cells (e.g., a somatic cell which hasbeen partially reprogrammed to an intermediate state between an inducedpluripotent stem cell and the somatic cell from which it was derived),multipotent cells, totipotent cells, a transdifferentiated version ofany of the foregoing cells, etc, as the invention is not intended to belimited in this manner). In some embodiments, the SC-β cells exhibit aresponse to multiple glucose challenges (e.g., at least one, at leasttwo, or at least three or more sequential glucose challenges). In someembodiments, the response resembles the response of endogenous islets(e.g., human islets) to multiple glucose challenges. In someembodiments, the morphology of the SC-β cell resembles the morphology ofan endogenous β cell. In some embodiments, the SC-β cell exhibits an invitro GSIS response that resembles the GSIS response of an endogenous 13cell. In some embodiments, the SC-β cell exhibits an in vivo GSISresponse that resembles the GSIS response of an endogenous β cell. Insome embodiments, the SC-β cell exhibits both an in vitro and in vivoGSIS response that resembles the GSIS response of an endogenous β cell.The GSIS response of the SC-β cell can be observed within two weeks oftransplantation of the SC-β cell into a host (e.g., a human or animal).In some embodiments, the SC-β cells package insulin into secretorygranules. In some embodiments, the SC-β cells exhibit encapsulatedcrystalline insulin granules. In some embodiments, the SC-β cellsexhibit a stimulation index of greater than 1. In some embodiments, theSC-β cells exhibit a stimulation index of greater than 1.1. In someembodiments, the SC-β cells exhibit a stimulation index of greater than2. In some embodiments, the SC-β cells exhibit cytokine-inducedapoptosis in response to cytokines. In some embodiments, insulinsecretion from the SC-β cells is enhanced in response to knownantidiabetic drugs (e.g., secretagogues). In some embodiments, the SC-βcells are monohormonal. In some embodiments, the SC-β cells do notabnormally co-express other hormones, such as glucagon, somatostatin orpancreatic polypeptide. In some embodiments, the SC-β cells exhibit alow rate of replication. In some embodiments, the SC-β cells increaseintracellular Ca2+ in response to glucose.

As used herein, the term “insulin producing cell” and its grammaticalequivalent refer to a cell differentiated from a pancreatic progenitor,or precursor thereof, which secretes insulin. An insulin-producing cellcan include pancreatic β cell as that term is described herein, as wellas pancreatic (β-like cells (e.g., insulin-positive, endocrine cells)that synthesize (e.g., transcribe the insulin gene, translate theproinsulin mRNA, and modify the proinsulin mRNA into the insulinprotein), express (e.g., manifest the phenotypic trait carried by theinsulin gene), or secrete (release insulin into the extracellular space)insulin in a constitutive or inducible manner. A population of insulinproducing cells e.g., produced by differentiating insulin-positive,endocrine cells or a precursor thereof into SC-β cells according to themethods of the present disclosure can be pancreatic β cell or (β-likecells (e.g., cells that have at least one, or at least two least two)characteristic of an endogenous β cell and exhibit a glucose stimulatedinsulin secretion (GSIS) response that resembles an endogenous adult βcell. The population of insulin-producing cells, e.g. produced by themethods as disclosed herein can comprise mature pancreatic β cell orSC-β cells, and can also contain non-insulin-producing cells (e.g.,cells of cell like phenotype with the exception they do not produce orsecrete insulin).

The terms “insulin-positive β-like cell,” “insulin-positive endocrinecell,” and their grammatical equivalents can refer to cells (e.g.,pancreatic endocrine cells) that displays at least one marker indicativeof a pancreatic β cell and also expresses insulin but lack a glucosestimulated insulin secretion (GSIS) response characteristic of anendogenous β cell.

The term “β cell marker” refers to, without limitation, proteins,peptides, nucleic acids, polymorphism of proteins and nucleic acids,splice variants, fragments of proteins or nucleic acids, elements, andother analyte which are specifically expressed or present in pancreaticβ cells. Exemplary β cell markers include, but are not limited to,pancreatic and duodenal homeobox 1 (Pdx1) polypeptide, insulin,c-peptide, amylin, E-cadherin, Hnf3β, PCI/3, B2, Nkx2.2, GLUT2, PC2,ZnT-8, ISL1, Pax6, Pax4, NeuroD, 1 Inflb, Hnf-6, Hnf-3beta, and MafA,and those described in Zhang et al., Diabetes. 50(10):2231-6 (2001). Insome embodiment, the β cell marker is a nuclear 3-cell marker. In someembodiments, the β cell marker is Pdx1 or PH3.

The term “pancreatic endocrine marker” can refer to without limitation,proteins, peptides, nucleic acids, polymorphism of proteins and nucleicacids, splice variants, fragments of proteins or nucleic acids,elements, and other analyte which are specifically expressed or presentin pancreatic endocrine cells. Exemplary pancreatic endocrine cellmarkers include, but are not limited to, Ngn-3, NeuroD and Islet-1.

The term “pancreatic progenitor,” “pancreatic endocrine progenitor,”“pancreatic precursor,” “pancreatic endocrine precursor” and theirgrammatical equivalents are used interchangeably herein and can refer toa stem cell which is capable of becoming a pancreatic hormone expressingcell capable of forming pancreatic endocrine cells, pancreatic exocrinecells or pancreatic duct cells. These cells are committed todifferentiating towards at least one type of pancreatic cell, e.g. βcells that produce insulin; a cells that produce glucagon; δcells (or Dcells) that produce somatostatin; and/or F cells that produce pancreaticpolypeptide. Such cells can express at least one of the followingmarkers: NGN3, NKX2.2, NeuroD, ISL-1, Pax4, Pax6, or ARX.

The term “Pdx1-positive pancreatic progenitor” as used herein can referto a cell which is a pancreatic endoderm (PE) cell which has thecapacity to differentiate into SC-β cells, such as pancreatic β cells. APdx1-positive pancreatic progenitor expresses the marker Pdx1. Othermarkers include, but are not limited to Cdcp1, or Ptf1a, or HNF6 orNRx2.2. The expression of Pdx1 may be assessed by any method known bythe skilled person such as immunochemistry using an anti-Pdx1 antibodyor quantitative RT-PCR. In some cases, a Pdx1-positive pancreaticprogenitor cell lacks expression of NKX6.1. In some cases, aPdx1-positive pancreatic progenitor cell can also be referred to asPdx1-positive, NKX6.1-negative pancreatic progenitor cell due to itslack of expression of NKX6.1. In some cases, the Pdx1-positivepancreatic progenitor cells can also be termed as “pancreatic foregutendoderm cells.”

The term “Pdx1-positive, NKX6-1-positive pancreatic progenitor” as usedherein can refer to a cell which is a pancreatic endoderm (PE) cellwhich has the capacity to differentiate into insulin-producing cells,such as pancreatic β cells. A Pdx1-positive, NKX6-1-positive pancreaticprogenitor expresses the markers Pdx1 and NKX6-1. Other markers include,but are not limited to Cdcp1, or Ptf1a, or HNF6 or NRx2.2. Theexpression of NKX6-1 may be assessed by any method known by the skilledperson such as immunochemistry using an anti-NKX6-1 antibody orquantitative RT-PCR. As used herein, the terms “NKX6.1” and “NKX6-1” areequivalent and interchangeable. In some cases, the Pdx1-positive,NKX6-1-positive pancreatic progenitor cells can also be termed as“pancreatic foregut precursor cells.”

The term “Ngn3-positive endocrine progenitor” as used herein can referto precursors of pancreatic endocrine cells expressing the transcriptionfactor Neurogenin-3 (Ngn3). Progenitor cells are more differentiatedthan multipotent stem cells and can differentiate into only few celltypes. In particular, Ngn3-positive endocrine progenitor cells have theability to differentiate into the five pancreatic endocrine cell types(α, β, δ, ε and PP). The expression of Ngn3 may be assessed by anymethod known by the skilled person such as immunochemistry using ananti-Ngn3 antibody or quantitative RT-PCR.

The terms “NeuroD” and “NeuroD1” are used interchangeably and identify aprotein expressed in pancreatic endocrine progenitor cells and the geneencoding it.

The term “selectable marker” refers to a gene, RNA, or protein that whenexpressed, confers upon cells a selectable phenotype, such as resistanceto a cytotoxic or cytostatic agent (e.g., antibiotic resistance),nutritional prototrophy, or expression of a particular protein that canbe used as a basis to distinguish cells that express the protein fromcells that do not. The term “selectable marker” as used herein can referto a gene or to an expression product of the gene, e.g., an encodedprotein. In some embodiments the selectable marker confers aproliferation and/or survival advantage on cells that express itrelative to cells that do not express it or that express it atsignificantly lower levels. Such proliferation and/or survival advantagetypically occurs when the cells are maintained under certain conditions,i.e., “selective conditions.” To ensure an effective selection, apopulation of cells can be maintained for a under conditions and for asufficient period of time such that cells that do not express the markerdo not proliferate and/or do not survive and are eliminated from thepopulation or their number is reduced to only a very small fraction ofthe population. The process of selecting cells that express a markerthat confers a proliferation and/or survival advantage by maintaining apopulation of cells under selective conditions so as to largely orcompletely eliminate cells that do not express the marker is referred toherein as “positive selection”, and the marker is said to be “useful forpositive selection”. Negative selection and markers useful for negativeselection are also of interest in certain of the methods describedherein. Expression of such markers confers a proliferation and/orsurvival disadvantage on cells that express the marker relative to cellsthat do not express the marker or express it at significantly lowerlevels (or, considered another way, cells that do not express the markerhave a proliferation and/or survival advantage relative to cells thatexpress the marker). Cells that express the marker can therefore belargely or completely eliminated from a population of cells whenmaintained in selective conditions for a sufficient period of time.

The term “epigenetics” refers to heritable changes in gene function thatdo not involve changes in the DNA sequence. Epigenetics most oftendenotes changes in a chromosome that affect gene activity andexpression, but can also be used to describe any heritable phenotypicchange that does not derive from a modification of the genome. Sucheffects on cellular and physiological phenotypic traits can result fromexternal or environmental factors, or be part of normal developmentalprogram. Epigenetics can also refer to functionally relevant changes tothe genome that do not involve a change in the nucleotide sequence.Examples of mechanisms that produce such changes are DNA methylation andhistone modification, each of which alters how genes are expressedwithout altering the underlying DNA sequence. Gene expression can becontrolled through the action of repressor proteins that attach tosilencer regions of the DNA. These epigenetic changes can last throughcell divisions for the duration of the cell's life, and can also lastfor multiple generations even though they do not involve changes in theunderlying DNA sequence of the organism. One example of an epigeneticchange in eukaryotic biology is the process of cellular differentiation.During morphogenesis, totipotent stem cells become the variouspluripotent cells, which in turn can become fully differentiated cells.

The term “epigenetic modifying compound” refers to a chemical compoundthat can make epigenetic changes genes, i.e., change gene expression(s)without changing DNA sequences. Epigenetic changes can help determinewhether genes are turned on or off and can influence the production ofproteins in certain cells, e.g., beta-cells. Epigenetic modifications,such as DNA methylation and histone modification, alter DNAaccessibility and chromatin structure, thereby regulating patterns ofgene expression. These processes are crucial to normal development anddifferentiation of distinct cell lineages in the adult organism. Theycan be modified by exogenous influences, and, as such, can contribute toor be the result of environmental alterations of phenotype orpathophenotype. Importantly, epigenetic modification has a crucial rolein the regulation of pluripotency genes, which become inactivated duringdifferentiation. Non-limiting exemplary epigenetic modifying compoundinclude a DNA methylation inhibitor, a histone acetyltransferaseinhibitor, a histone deacetylase inhibitor, a histone methyltransferaseinhibitor, a bromodomain inhibitor, or any combination thereof.

The term “differentiated cell” or its grammatical equivalents is meantany primary cell that is not, in its native form, pluripotent as thatterm is defined herein. Stated another way, the term “differentiatedcell” can refer to a cell of a more specialized cell type derived from acell of a less specialized cell type (e.g., a stem cell such as aninduced pluripotent stem cell) in a cellular differentiation process.Without wishing to be limited to theory, a pluripotent stem cell in thecourse of normal ontogeny can differentiate first to an endoderm cellthat is capable of forming pancreas cells and other endoderm cell types.Further differentiation of an endoderm cell leads to the pancreaticpathway, where ^(˜)98% of the cells become exocrine, ductular, or matrixcells, and ˜2% become endocrine cells. Early endocrine cells are isletprogenitors, which can then differentiate further into insulin-producingcells (e.g. functional endocrine cells) which secrete insulin, glucagon,somatostatin, or pancreatic polypeptide. Endoderm cells can also bedifferentiate into other cells of endodermal origin, e.g. lung, liver,intestine, thymus etc.

As used herein, the term “somatic cell” can refer to any cells formingthe body of an organism, as opposed to germline cells. In mammals,germline cells (also known as “gametes”) are the spermatozoa and ovawhich fuse during fertilization to produce a cell called a zygote, fromwhich the entire mammalian embryo develops. Every other cell type in themammalian body—apart from the sperm and ova, the cells from which theyare made (gametocytes) and undifferentiated stem cells—is a somaticcell: internal organs, skin, bones, blood, and connective tissue are allmade up of somatic cells. In some embodiments the somatic cell is a“non-embryonic somatic cell”, by which is meant a somatic cell that isnot present in or obtained from an embryo and does not result fromproliferation of such a cell in vitro. In some embodiments the somaticcell is an “adult somatic cell”, by which is meant a cell that ispresent in or obtained from an organism other than an embryo or a fetusor results from proliferation of such a cell in vitro. Unless otherwiseindicated the methods for converting at least one insulin-positiveendocrine cell or precursor thereof to an insulin-producing, glucoseresponsive cell can be performed both in vivo and in vitro (where invivo is practiced when at least one insulin-positive endocrine cell orprecursor thereof are present within a subject, and where in vitro ispracticed using an isolated at least one insulin-positive endocrine cellor precursor thereof maintained in culture).

As used herein, the term “adult cell” can refer to a cell foundthroughout the body after embryonic development.

The term “endoderm cell” as used herein can refer to a cell which isfrom one of the three primary germ cell layers in the very early embryo(the other two germ cell layers are the mesoderm and ectoderm). Theendoderm is the innermost of the three layers. An endoderm celldifferentiates to give rise first to the embryonic gut and then to thelinings of the respiratory and digestive tracts (e.g. the intestine),the liver and the pancreas.

The term “a cell of endoderm origin” as used herein can refer to anycell which has developed or differentiated from an endoderm cell. Forexample, a cell of endoderm origin includes cells of the liver, lung,pancreas, thymus, intestine, stomach and thyroid. Without wishing to bebound by theory, liver and pancreas progenitors (also referred to aspancreatic progenitors) are develop from endoderm cells in the embryonicforegut. Shortly after their specification, liver and pancreasprogenitors rapidly acquire markedly different cellular functions andregenerative capacities. These changes are elicited by inductive signalsand genetic regulatory factors that are highly conserved amongvertebrates. Interest in the development and regeneration of the organshas been fueled by the intense need for hepatocytes and pancreatic βcells in the therapeutic treatment of liver failure and type I diabetes.Studies in diverse model organisms and humans have revealedevolutionarily conserved inductive signals and transcription factornetworks that elicit the differentiation of liver and pancreatic cellsand provide guidance for how to promote hepatocyte and β celldifferentiation from diverse stem and progenitor cell types.

The term “definitive endoderm” as used herein can refer to a celldifferentiated from an endoderm cell and which can be differentiatedinto a SC-β cell (e.g., a pancreatic β cell). A definitive endoderm cellexpresses the marker Sox17. Other markers characteristic of definitiveendoderm cells include, but are not limited to MIXL2, GATA4, HNF3b, GSC,FGF17, VWF, CALCR, FOXQ1, CXCR4, Cerberus, OTX2, goosecoid, C-Kit, CD99,CMKOR1 and CRIP1. In particular, definitive endoderm cells hereinexpress Sox17 and in some embodiments Sox17 and HNF3B, and do notexpress significant levels of GATA4, SPARC, APF or DAB. Definitiveendoderm cells are not positive for the marker Pdx1 (e.g. they arePdx1-negative). Definitive endoderm cells have the capacity todifferentiate into cells including those of the liver, lung, pancreas,thymus, intestine, stomach and thyroid. The expression of Sox17 andother markers of definitive endoderm may be assessed by any method knownby the skilled person such as immunochemistry, e.g., using an anti-Sox17antibody, or quantitative RT-PCR.

The term “pancreatic endoderm” can refer to a cell of endoderm originwhich is capable of differentiating into multiple pancreatic lineages,including pancreatic β cells, but no longer has the capacity todifferentiate into non-pancreatic lineages.

The term “primitive gut tube cell” or “gut tube cell” as used herein canrefer to a cell differentiated from an endoderm cell and which can bedifferentiated into a SC-β cell (e.g., a pancreatic β cell). A primitivegut tube cell expresses at least one of the following markers: HNP1-β,HNF3-β or HNF4-α. Primitive gut tube cells have the capacity todifferentiate into cells including those of the lung, liver, pancreas,stomach, and intestine. The expression of HNF1-β and other markers ofprimitive gut tube may be assessed by any method known by the skilledperson such as immunochemistry, e.g., using an anti-HNF1-β antibody.

The term “stem cell” as used herein, can refer to an undifferentiatedcell which is capable of proliferation and giving rise to moreprogenitor cells having the ability to generate a large number of mothercells that can in turn give rise to differentiated, or differentiabledaughter cells. The daughter cells themselves can be induced toproliferate and produce progeny that subsequently differentiate into oneor more mature cell types, while also retaining one or more cells withparental developmental potential. The term “stem cell” can refer to asubset of progenitors that have the capacity or potential, underparticular circumstances, to differentiate to a more specialized ordifferentiated phenotype, and which retains the capacity, under certaincircumstances, to proliferate without substantially differentiating. Inone embodiment, the term stem cell refers generally to a naturallyoccurring mother cell whose descendants (progeny) specialize, often indifferent directions, by differentiation, e.g., by acquiring completelyindividual characters, as occurs in progressive diversification ofembryonic cells and tissues. Cellular differentiation is a complexprocess typically occurring through many cell divisions. Adifferentiated cell may derive from a multipotent cell which itself isderived from a multipotent cell, and so on. While each of thesemultipotent cells may be considered stem cells, the range of cell typeseach can give rise to may vary considerably. Some differentiated cellsalso have the capacity to give rise to cells of greater developmentalpotential. Such capacity may be natural or may be induced artificiallyupon treatment with various factors. In many biological instances, stemcells are also “multipotent” because they can produce progeny of morethan one distinct cell type, but this is not required for “stem-ness.”Self-renewal is the other classical part of the stem cell definition,and it is essential as used in this document. In theory, self-renewalcan occur by either of two major mechanisms. Stem cells may divideasymmetrically, with one daughter retaining the stem state and the otherdaughter expressing some distinct other specific function and phenotype.Alternatively, some of the stem cells in a population can dividesymmetrically into two stems, thus maintaining some stem cells in thepopulation as a whole, while other cells in the population give rise todifferentiated progeny only. Formally, it is possible that cells thatbegin as stem cells might proceed toward a differentiated phenotype, butthen “reverse” and re-express the stem cell phenotype, a term oftenreferred to as “dedifferentiation” or “reprogramming” or“retro-differentiation” by persons of ordinary skill in the art. As usedherein, the term “pluripotent stem cell” includes embryonic stem cells,induced pluripotent stem cells, placental stem cells, etc.

The term “pluripotent” as used herein can refer to a cell with thecapacity, under different conditions, to differentiate to more than onedifferentiated cell type, and preferably to differentiate to cell typescharacteristic of all three germ cell layers. Pluripotent cells arecharacterized primarily by their ability to differentiate to more thanone cell type, preferably to all three germ layers, using, for example,a nude mouse teratoma formation assay. Pluripotency is also evidenced bythe expression of embryonic stem (ES) cell markers, although thepreferred test for pluripotency is the demonstration of the capacity todifferentiate into cells of each of the three germ layers. It should benoted that simply culturing such cells does not, on its own, render thempluripotent. Reprogrammed pluripotent cells (e.g. iPS cells as that termis defined herein) also have the characteristic of the capacity ofextended passaging without loss of growth potential, relative to primarycell parents, which generally have capacity for only a limited number ofdivisions in culture.

As used herein, the terms “iPS cell” and “induced pluripotent stem cell”are used interchangeably and can refer to a pluripotent stem cellartificially derived (e.g., induced or by complete reversal) from anon-pluripotent cell, typically an adult somatic cell, for example, byinducing a forced expression of one or more genes.

The term “phenotype” can refer to one or a number of total biologicalcharacteristics that define the cell or organism under a particular setof environmental conditions and factors, regardless of the actualgenotype.

The terms “subject,” “patient,” or “individual” are used interchangeablyherein, and can refer to an animal, for example, a human from whom cellscan be obtained and/or to whom treatment, including prophylactictreatment, with the cells as described herein, is provided. Fortreatment of those infections, conditions or disease states which arespecific for a specific animal such as a human subject, the term subjectcan refer to that specific animal. The “non-human animals” and“non-human mammals” as used interchangeably herein, includes mammalssuch as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, andnon-human primates. The term “subject” also encompasses any vertebrateincluding but not limited to mammals, reptiles, amphibians and fish.However, advantageously, the subject is a mammal such as a human, orother mammals such as a domesticated mammal, e.g, dog, cat, horse, andthe like, or production mammal, e.g. cow, sheep, pig, and the like.“Patient in need thereof” or “subject in need thereof” is referred toherein as a patient diagnosed with or suspected of having a disease ordisorder, for instance, but not restricted to diabetes.

“Administering” used herein can refer to providing one or morecompositions described herein to a patient or a subject. By way ofexample and not limitation, composition administration, e.g., injection,can be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.)injection, intradermal (i.d.) injection, intraperitoneal (i.p.)injection, or intramuscular (i.m.) injection. One or more such routescan be employed. Parenteral administration can be, for example, by bolusinjection or by gradual perfusion over time. Alternatively, orconcurrently, administration can be by the oral route. Additionally,administration can also be by surgical deposition of a bolus or pelletof cells, or positioning of a medical device. In an embodiment, acomposition of the present disclosure can comprise engineered cells orhost cells expressing nucleic acid sequences described herein, or avector comprising at least one nucleic acid sequence described herein,in an amount that is effective to treat or prevent proliferativedisorders. A pharmaceutical composition can comprise the cell populationas described herein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions can comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

The terms “treat,” “treating,” “treatment,” and their grammaticalequivalents, as applied to an isolated cell, include subjecting the cellto any kind of process or condition or performing any kind ofmanipulation or procedure on the cell. As applied to a subject, theterms refer to providing medical or surgical attention, care, ormanagement to an individual. The individual is usually ill or injured,or at increased risk of becoming ill relative to an average member ofthe population and in need of such attention, care, or management.

As used herein, the term “treating” and “treatment” can refer toadministering to a subject an effective amount of a composition so thatthe subject as a reduction in at least one symptom of the disease or animprovement in the disease, for example, beneficial or desired clinicalresults. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation of one or moresymptoms, diminishment of extent of disease, stabilized (e.g., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. Treating canrefer to prolonging survival as compared to expected survival if notreceiving treatment. Thus, one of skill in the art realizes that atreatment may improve the disease condition, but may not be a completecure for the disease. As used herein, the term “treatment” includesprophylaxis. Alternatively, treatment is “effective” if the progressionof a disease is reduced or halted. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already diagnosed with acardiac condition, as well as those likely to develop a cardiaccondition due to genetic susceptibility or other factors such as weight,diet and health.

The term “therapeutically effective amount”, therapeutic amount”, or itsgrammatical equivalents can refer to an amount effective, at dosages andfor periods of time necessary, to achieve a desired therapeutic result.The therapeutically effective amount can vary according to factors suchas the disease state, age, sex, and weight of the individual and theability of a composition described herein to elicit a desired responsein one or more subjects. The precise amount of the compositions of thepresent disclosure to be administered can be determined by a physicianwith consideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject).

Alternatively, the pharmacologic and/or physiologic effect ofadministration of one or more compositions described herein to a patientor a subject of can be “prophylactic,” e.g., the effect completely orpartially prevents a disease or symptom thereof. A “prophylacticallyeffective amount” can refer to an amount effective, at dosages and forperiods of time necessary, to achieve a desired prophylactic result(e.g., prevention of disease onset).

Some numerical values disclosed throughout are referred to as, forexample, “X is at least or at least about 100; or 200 [or any numericalnumber].” This numerical value includes the number itself and all of thefollowing:

i) X is at least 100;ii) X is at least 200;iii) X is at least about 100; andiv) X is at least about 200.

All these different combinations are contemplated by the numericalvalues disclosed throughout. All disclosed numerical values should beinterpreted in this manner, whether it refers to an administration of atherapeutic agent or referring to days, months, years, weight, dosageamounts, etc., unless otherwise specifically indicated to the contrary.

The ranges disclosed throughout are sometimes referred to as, forexample, “X is administered on or on about day 1 to 2; or 2 to 3 [or anynumerical range].” This range includes the numbers themselves (e.g., theendpoints of the range) and all of the following:

i) X being administered on between day 1 and day 2;ii) X being administered on between day 2 and day 3;iii) X being administered on between about day 1 and day 2;iv) X being administered on between about day 2 and day 3;v) X being administered on between day 1 and about day 2;vi) X being administered on between day 2 and about day 3;vii) X being administered on between about day 1 and about day 2; andviii) X being administered on between about day 2 and about day 3.

All these different combinations are contemplated by the rangesdisclosed throughout. All disclosed ranges should be interpreted in thismanner, whether it refers to an administration of a therapeutic agent orreferring to days, months, years, weight, dosage amounts, etc., unlessotherwise specifically indicated to the contrary.

I. Overview

In aspects, the present disclosure provides compositions and methods ofdifferentiating pancreatic progenitor cells. The compositions andmethods provided herein can offer pancreatic β cells, cell populations,or cell clusters that have high purity of pancreatic β cells, highinsulin content, and superior glucose-dependent insulin secretionresponse that can resemble native pancreatic β cells or nativepancreatic islets.

In some aspects, provided herein is a method that comprises contacting apopulation of pancreatic progenitor cells or precursors thereof with anepigenetic modifying compound, wherein the contacting results in apopulation of endocrine cells with an increased proportion ofchromogranin A-positive (CHGA+) cells or an increased proportion ofC-peptide-positive and NKX6.1-positive (C-PEP+, NKX6.1+) cells ascompared to a corresponding population of endocrine cells which is notcontacted with the epigenetic modifying compound.

In some aspects, the present disclosure provides a method comprising:contacting a population of pancreatic progenitor cells or precursorsthereof with an epigenetic modifying compound, wherein the contactingresults in a population of endocrine cells with a reduced proportion ofcells expressing VMAT or Cdx2 as compared to a corresponding populationof endocrine cells which is not contacted with the epigenetic modifyingcompound.

In some aspects, the present disclosure provides a compositioncomprising a cell population, wherein the cell population comprises: (a)at least about 20% cells expressing C-peptide and NKX6.1; (b) at leastabout 60% cells expressing CHGA; (c) at most about 20% cells expressingCdx2; or (d) at most about 45% cells expressing VMAT1, as measured byflow cytometry. In some cases, the composition comprises: (a) at leastabout 20% cells expressing C-peptide and NKX6.1;

(b) at least about 60% cells expressing CHGA; and (c) at most about 20%cells expressing Cdx2, as measured by flow cytometry. In some cases, thecomposition also comprises at most about 45% cells expressing VMAT1, asmeasured by flow cytometry. In some cases, the composition furthercomprises an epigenetic modifying compound.

In some aspects, the present disclosure provides a compositioncomprising a cell population that comprises at least about 30%ISL1-positive, NKX6.1-positive cells and at most about 20%ISL1-negative, NKX6.1-negative cells, as measured by flow cytometry. Thecomposition of claim 34, wherein the cell population comprises at leastabout 35% ISL1-positive, NKX6.1-positive cells. In some cases, the cellpopulation comprises at least about 40% ISL1-positive, NKX6.1-positivecells. In some cases, the cell population comprises at most about 15%ISL1-negative, NKX6.1-negative cells. In some cases, the compositionfurther comprises an epigenetic modifying compound.

In some aspects, the present disclosure provides a composition thatcomprises a pancreatic progenitor cell, and at least one of a histonedeacetylase (HDAC) inhibitor or a histone methyltransferase inhibitor.In some aspects, the present disclosure provides a method comprising:contacting a cell population comprising pancreatic progenitor cells orprecursors thereof with a histone methyltransferase inhibitor andgenerating a cell population comprising endocrine cells; and maturingthe cell population comprising endocrine cells to obtain at least onepancreatic β cell that exhibits an in vitro glucose-stimulated insulinsecretion response to a glucose challenge. In some cases, the maturationstep is conducted after the cell population comprising endocrine cellsis implanted into a subject in vivo. In some cases, the maturation stepis performed in vitro.

In some aspects, the present disclosure provides a method, comprising:(a) contacting a population of Pdx1-negative, NKX6.1-negative primitivegut tube cells with a bone morphogenetic protein (BMP) signaling pathwayinhibitor and a growth factor from transformation growth factor β(TGF-β) superfamily, thereby generating a cell population that comprisesPdx1-positive, NKX6.1-positive pancreatic progenitor cells; and (b)contacting the cell population comprising the Pdx1-positive,NKX6.1-positive pancreatic progenitor cells with an epigenetic modifyingcompound and generating a cell population comprising endocrine cells.

In some cases, the methods provided herein comprise A method,comprising: (a) differentiating pluripotent stem cells in a populationinto definitive endoderm cells by contacting the pluripotent stem cellswith a growth factor from TGF-β superfamily and a WNT signaling pathwayactivator; (b) differentiating at least some of the definitive endodermcells into primitive gut tube cells by contacting the definitiveendoderm cells with a growth factor from FGF family; (c) differentiatingat least some of the primitive gut tube cells into Pdx1-positivepancreatic progenitor cells by contacting the primitive gut tube cellswith a ROCK inhibitor, a growth factor from FGF family, a BMP signalingpathway inhibitor, a PKC activator, a retinoic acid signaling pathwayactivator, a SHH pathway inhibitor, and a growth factor from TGF-βsuperfamily; (d) differentiating at least some of the Pdx1-positivepancreatic progenitor cells into Pdx1-positive, NKX6.1-positivepancreatic progenitor cells by contacting the Pdx1-positive pancreaticprogenitor cells with a ROCK inhibitor, a growth factor from TGFβsuperfamily, a growth factor from FGF family, a RA signaling pathwayactivator, and a SHH pathway inhibitor; and (e) differentiating at leastsome of the Pdx1-positive, NKX6.1-positive pancreatic progenitor cellsinto a cell population comprising at least one NKX6.1+ and C-peptide+cell by contacting the Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells with a TGF-β signaling pathway inhibitor, a growthfactor from EGF family, a RA signaling pathway activator, a SHH pathwayinhibitor, a TH signaling pathway activator, a γ-secretase inhibitor, aprotein kinase inhibitor, a ROCK inhibitor, a BMP signaling pathwayinhibitor, and an epigenetic modifying compound.

In some aspects, the present disclosure provides a method of irradiatingcells to reduce proliferation. In some cases, the methods compriseexposing an in vitro cell population comprising endocrine cells toirradiation at a dose of about 100 rads to about 100,000 rads for a timeperiod of about 1 min to about 60 min.

In some cases, the methods of reducing cell proliferation compriseexposing to irradiation a cell population comprising stem cells,definitive endoderm cells, primitive gut tube cells, pancreaticprogenitor cells, or endocrine cells, wherein the irradiation results ina cell population that has reduced proliferative capability as comparedto a corresponding cell population that is not subject to irradiation.

II. Method of Generating Endocrine Cells

In aspects, the present disclosure relates to compositions and methodsof generating endocrine cells from pancreatic progenitor cells orprecursors. Certain exemplary detailed protocols of generating endocrinecells the stem cells to provide at least one SC-β cell are described inU.S. Patent Application Publication No. US20150240212 and US20150218522,each of which is herein incorporated by reference in its entirety.

In some cases, a method for generating a first population of endocrinecells comprises contacting a population of pancreatic progenitor cellsor precursors thereof with a first composition comprising at least oneepigenetic modifying compound to generate the first population ofendocrine cells, wherein a reduced proportion of cells of the firstpopulation of endocrine cells express VMAT⁺ or Cdx2⁺ as compared to asecond population of endocrine cells generated using a secondcomposition that lacks the at least one epigenetic modifying compound.In some embodiments, an epigenetic modifying compound is added at stage5 (FIGS. 5-6 ), which can induce endocrine cells to shift the proportionof endocrine cells in following ways: (1) reducing an endocrinepopulation marked by VMAT1 marker (FIG. 19 ) or by Cdx2; (2) increasingthe proportion of cells fated to the alpha cell and other non-beta cellpancreatic islet cell fates (FIG. 20 ); and (3) increasing theproportion of beta cells in the composition (FIG. 8 ).

In some embodiments, the first population of endocrine cells is VMAT+and INS−. In some embodiments, the population of pancreatic progenitorcells differentiate into a population of PH cells. In some embodiments,an increased proportion of cells of the first population of endocrinecells are NKX6.1⁻ or ChromA⁺ as compared to the second population ofendocrine cells generated using the second composition that lacks theepigenetic modifying compound. In some embodiments, the increasedproportion of cells is NKX6.1⁻ and ChromA⁺. In some embodiments, thefirst population of pancreatic progenitor cells differentiates into apopulation of β cells. In some embodiments, the β cells are stem-cellderived β (SC-β) cells. In some embodiments, the β cells express C-PEPand NKX6-1. In some embodiments, the β cells exhibit an in vitroglucose-stimulated insulin secretion response to a glucose challenge. Insome embodiments, the methods described herein are performed in vitro.

In some cases, the first composition and the second composition compriseat least one of i) a SHH pathway inhibitor, ii) a RA signaling pathwayactivator, iii) a γ-secretase inhibitor, iv) at least one growth factorfrom the epidermal growth factor (EGF) family, v) a protein kinaseinhibitor, vi) a BMP signaling pathway inhibitor, vii) a TGF-β signalingpathway inhibitor, viii) a thyroid hormone signaling pathway agonist, orix) a ROCK inhibitor. In some embodiments, the first composition and thesecond composition comprise at least one of betacellulin, thiazovinin,retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, staurosporine, or anycombination thereof. In some cases, the first composition comprises andthe second composition both comprise betacellulin, thiazovinin, retinoicacid, SANT1, XXI, Alk5i II, GC-1, LDN, and staurosporine.

Provided herein is a method of generating an endocrine cell comprisingcontacting a pancreatic progenitor cell or precursor thereof with ahistone methyltransferase inhibitor, wherein the contacting induces thepancreatic progenitor cell to differentiate into the endocrine cell. Insome cases, the histone methyltransferase inhibitor comprises DZNep. Insome cases, the method further comprises contacting the pancreaticprogenitor cell or precursor thereof with at least one of i) a SHHpathway inhibitor, ii) a RA signaling pathway activator, iii) aγ-secretase inhibitor, iv) at least one growth factor from the epidermalgrowth factor (EGF) family, v) a protein kinase inhibitor, vi) a BMPsignaling pathway inhibitor, vii) a TGF-β signaling pathway inhibitor,viii) a thyroid hormone signaling pathway agonist, or ix) a ROCKinhibitor.

Provided herein is a method of generating an endocrine cell comprisingcontacting a pancreatic progenitor cell or precursor thereof with ahistone deacetylase (HDAC) inhibitor and a histone methyltransferaseinhibitor, wherein the contacting induces the pancreatic progenitor cellto differentiate into the endocrine cell. In some cases, the methodfurther comprises contacting the pancreatic progenitor cell or precursorthere of with at least one of i) a SHH pathway inhibitor, ii) a RAsignaling pathway activator, iii) a γ-secretase inhibitor, iv) at leastone growth factor from the epidermal growth factor (EGF) family, v) aprotein kinase inhibitor, vi) a BMP signaling pathway inhibitor, vii) aTGF-β signaling pathway inhibitor, viii) a thyroid hormone signalingpathway agonist, or ix) a ROCK inhibitor.

In some embodiments, the endocrine cell differentiates into a β cell. Insome embodiments, the β cell is a stem-cell derived β (SC-β) cell. Insome embodiments, the β cell expresses C-PEP and NKX6-1. In someembodiments, the β cells exhibit an in vitro glucose-stimulated insulinsecretion response to a glucose challenge. In some embodiments, the HDACinhibitor is a Class I HDAC inhibitor, a Class II HDAC inhibitor, or acombination thereof.

Provided herein is a method of generating an endocrine cell comprisingcontacting a pancreatic progenitor cell or precursor thereof with ahistone deacetylase (HDAC) inhibitor, wherein the HDAC inhibitor isKD5170. In some embodiments, the method further comprises contractingthe pancreatic progenitor cell with a histone methyltransferaseinhibitor. In some embodiments, the endocrine cell expresses CHGA. Insome embodiments, the endocrine cell differentiates into a β cell. Insome embodiments, the β cell is a stem-cell derived β (SC-β) cell. Insome embodiments, the β cell expresses C-PEP and NKX6-1. In someembodiments, the β cell exhibits an in vitro glucose-stimulated insulinsecretion response to a glucose challenge.

Also provided herein is a composition that comprises a population ofcells and a culturing medium, wherein the cells comprise a pancreaticprogenitor cell, and wherein the culturing medium comprises a histonedeacetylase (HDAC) inhibitor and a histone methyltransferase inhibitor.In some embodiments, the pancreatic progenitor cell when contacted withthe culturing medium is induced to differentiate into an endocrine cell.

Non-limiting exemplary epigenetic modifying compound include a DNAmethylation inhibitor, a histone acetyltransferase inhibitor, a histonedeacetylase inhibitor, a histone methyltransferase inhibitor, abromodomain inhibitor, or any combination thereof.

In an embodiment, the histone methyltransferase inhibitor is aninhibitor of enhancer of zeste homolog 2 (EZH2). EZH2 is ahistone-lysine N-methyltransferase enzyme. Non-limiting examples of anEZH2 inhibitor include 3-deazaneplanocin A (DZNep), EPZ6438, EPZ005687(an S-adenosylmethionine (SAM) competitive inhibitor), EI1, GSK126, andUNC1999. DZNep inhibits the hydrolysis of S-adenosyl-L-homocysteine(SAH), which is a product-based inhibitor of all proteinmethyltransferases, leading to increased cellular concentrations of SAHwhich in turn inhibits EZH2. DZNep is not specific to EZH2 and alsoinhibits other DNA methyltransferases. GSK126 is a SAM-competitive EZH2inhibitor that has 150-fold selectivity over EZH1. UNC1999 is ananalogue of GSK126, and it is less selective than its counterpartGSK126.

In an embodiment, the histone methyltransferase inhibitor is DZNep. Inan embodiment, the HDAC inhibitor is a class I HDAC inhibitor, a classII HDAC inhibitor, or a combination thereof. In an embodiment, the HDACinhibitor is KD5170 (mercaptoketone-based HDAC inhibitor), MC1568 (classIIa HDAC inhibitor), TMP195 (class IIa HDAC inhibitor), or anycombination thereof. In some embodiments, the HDAC inhibitor isvorinostat, romidepsin (Istodax), chidamide, panobinostat (farydak),belinostat (PXD101), panobinostat (LBH589), valproic acid, mocetinostat(MGCD0103), abexinostat (PCI-24781), entinostat (MS-275), SB939,resminostat (4SC-201), givinostat (ITF2357), quisinostat (JNJ-26481585),HBI-8000, (a benzamide HDI), kevetrin, CUDC-101, AR-42, CHR-2845,CHR-3996, 4SC-202, CG200745, ACY-1215, ME-344, sulforaphane, or anyvariant thereof.

In some cases, the concentration of the histone methyltransferaseinhibitor (e.g., DZNep) can be from or from about 0.01 to 10 μM. Forexample, the concentration of the histone methyltransferase inhibitor(e.g., DZNep) can be about 0.01 to 1, 0.1 to 1, 0.25 to 1, 0.5 to 1, 1to 5; or 1 to 10 μM. The concentration of the histone methyltransferaseinhibitor (e.g., DZNep) can be less than about: 5, 4, 3, 2, 1.9, 1.8,1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3,0.2, 0.1, 0.05, or 0.01 μM.

In some embodiment, the method comprises contacting the pancreaticprogenitor cells or precursors with the first or second composition forat least 1 day, at least 2 days, at least 3 days, at least 4 days, atleast 5 days, at least 6 days, at least 7 days, at least 8 days, atleast 9 days, at least 10 days, at least 11 days, at least 12 days, atleast 13 days, at least 14 days, at least 15 days, at least 16 days, atleast 17 days, at least 18 days, at least 19 days, or at least 20 days.In some embodiment, the method comprises contacting the pancreaticprogenitor cells or precursors with the first or second composition forabout 1 day, about 2 days, about 3 days, about 4 days, about 5 daysabout 6 days, about 7 days, about 8 days, about 9 days, about 10 days,about 11 days, about 12 days, about 13 days, about 14 days, about 15days, about 16 days, about 17 days, about 18 days, about 19 days, orabout 20 days.

In some embodiments, the pancreatic progenitor cell expresses at leastone of PDX1 and NKX6.1. In some embodiments, the pancreatic progenitorcell expresses both PDX1 and NKX6.1. In some embodiments, the endocrinecell expresses CHGA.

Epigenetics Modifications

Epigenetics can refer to heritable alterations that are not due tochanges in DNA sequence. Rather, epigenetic modifications, such as DNAmethylation and histone modification, can alter DNA accessibility andchromatin structure, thereby regulating patterns of gene expression.These processes can be crucial to normal development and differentiationof distinct cell lineages in the adult organism. They can be modified byexogenous influences, and, as such, can contribute to or be the resultof environmental alterations of phenotype or pathophenotype.Importantly, epigenetic programming can have a crucial role in theregulation of pluripotency genes, which become inactivated duringdifferentiation.

Chromatin is the complex of chromosomal DNA associated with proteins inthe nucleus. DNA in chromatin is packaged around histone proteins, inunits referred to as nucleosomes. A nucleosome can have 147 base-pairsof DNA associated with an octomeric core of histone proteins, consistingof two H3-H4 histone dimers surrounded by two H2A-H2B dimers. N-terminalhistone tails can protrude from nucleosomes into the nuclear lumen. H1histone can associate with the linker DNA located between thenucleosomes. Nucleosome spacing determines chromatin structure which canbe broadly divided into heterochromatin and euchromatin. Chromatinstructure and gene accessibility to transcriptional machinery can beregulated by modifications to both DNA and histone tails.

In differentiated mammalian cells, the principal epigenetic modificationfound in DNA can be that of covalent attachment of a methyl group to theC5 position of cytosine residues in CpG dinucleotide sequences (DNAmethylation). In undifferentiated stem cells, cytosines, other thanthose in CpG, can be methylated, as well and that methylation of non-CpGcytosines can be crucial for gene regulation in embryonic stem cells inparticular. CpG methylation can be, however, an important mechanism toensure the repression of transcription of repeat elements andtransposons, and also can play a crucial role in imprinting andX-chromosome inactivation. Transcriptional gene silencing by CpGmethylation can also restrict the expression of some tissue-specificgenes during development and differentiation by repressing them innon-expressing cells.

CpG methylation can suppress transcription by several mechanisms. Thepresence of the methyl group at a specific CpG may directly block DNArecognition and binding by some transcription factors. Alternatively,other factors may preferentially bind to methylated DNA, blockingtranscription factor access. For example MeCP2 and other family memberscan bind to methyl CpG and contribute to transcriptional repression bythe recruitment of histone-modifying proteins, such as histonedeacetylases (HDAC). Subsequently, histone deacetylation can promotechromatin condensation, further repressing transcription. This sequenceof events illustrates how DNA methylation and certain histonemodifications function together to contribute to the transcriptional onor off state of genes subject to epigenetic modification.

A family of DNA methyltransferase enzymes (DNMTs) is involved in de novoDNA methylation and its maintenance. During embryogenesis, de novomethylation can be carried out by DNMT3A and DNMT3B 15. The ubiquitouslyexpressed DNMT1 can be predominantly responsible for maintainingcellular levels of CpG methylation. DNMT1 can function in a complex torecognize hemi-methylated DNA and to add methyl groups to thenon-methylated daughter strand formed during replication. The basepairing of CpG can allow for the reciprocal maintenance of methylationduring subsequent replication cycles. In this manner, a non-genetictrait (DNA methylation) can be passed from cell to cell and, with it,the contextual effects on gene expression. Thus, methylation can beconsidered a long-term, relatively stable, epigenetic trait, the effectsof which can contribute to maintaining the cellular phenotype.

DNA methylation can promote the persistence of certain histone states,such as deacetylation, thus providing a mechanism for perpetuatingpost-translational histone modifications. Histones can bepost-translationally modified to restructure chromatin in many ways,including phosphorylation, ubiquitination, acetylation, and methylation.Of these histone modifications, histone acetylation, at the ε-aminogroup of lysine residues in H3 and H4 tails, can be highly consistentlyassociated with promoting transcription. Acetylated, open-chromatinstructure can also allow access of transcriptional repressors. Forexample, some bromodomain-containing factors, such as BRG1 and Brd4,target to acetylated histones where they can mediate the formation ofrepressor (or activator) complexes. Acetylation can be targeted toregions of chromatin by the recognition and binding of DNAsequence-specific transcription factors that recruit one of a growingfamily of histone acetyl transferase (HAT) cofactors such as CREBbinding protein (CBP), and p300, MYST, and GNAT.

Deacetylation of histones can correlate with CpG methylation and theinactive state of chromatin. There are 4 classes of histone deacetylaseenzymes (HDACs), with members capable of deacetylation of histonesand/or other protein targets. These regulatory proteins can bethemselves subject to regulation by acetylation, phosphorylation, andsumoylation, which can affect their function, subcellular distribution,and protein-protein associations. Interactions with sequence-specificDNA binding proteins and co-repressor complexes can target certain HDACproteins to histones in a gene-specific manner.

Most histone lysine methyltransferases can have a SET homology domain, avast family of proteins that can be grouped into 7 subfamilies based ontheir structural similarities. SET1 family members can specificallyfoster active chromatin by methylating H3K4. Other histone lysinemethyltransferase families can methylate several histone targets. Inaddition, some of these methyl transferases can have additional domainsthat specify binding to methylated DNA or to other proteins, such as CBP39. HDAC proteins can comprise a family of 18 members in humans and areseparated into four classes based on their size, cellular localization,number of catalytic active sites, and homology to yeast HDAC proteins.Class I includes HDAC1, HDAC2, HDAC3, and HDAC8. Class II consists ofsix HDAC proteins that are further divided into two subclasses. ClassIIa includes HDAC4, HDAC5, HDAC7, and HDAC9, which each contains asingle catalytic active site. Class IIb includes HDAC6 and HDAC10, whichboth contain two active sites, although only HDAC6 has two catalyticallycompetent active sites. HDAC11 is the sole member of class IV, based onphylogenetic analysis. Class I, II, and IV HDAC proteins can operate bya metal ion-dependent mechanism, as indicated by crystallographicanalysis. In contrast, class III HDAC proteins, referred to as sirtuins(SIRT1-7), can operate by a NAD+-dependent mechanism unrelated to theother HDAC proteins. In an embodiment, HDAC inhibitors of HDAC proteinsinduce cell differentiation. In an embodiment, HDAC inhibitorsupregulate crucial genes associated with cell differentiation.

Epigenetic Modifying Compounds

The term “epigenetic modifying compound” can refer to a chemicalcompound that can make epigenetic changes genes, i.e., change geneexpression(s) without changing DNA sequences. Epigenetic changes canhelp determine whether genes are turned on or off and can influence theproduction of proteins in certain cells, e.g., beta-cells. Epigeneticmodifications, such as DNA methylation and histone modification, canalter DNA accessibility and chromatin structure, thereby regulatingpatterns of gene expression. These processes can be crucial to normaldevelopment and differentiation of distinct cell lineages in the adultorganism. They can be modified by exogenous influences, and, as such,can contribute to or be the result of environmental alterations ofphenotype or pathophenotype. Importantly, epigenetic modification canhave a crucial role in the regulation of pluripotency genes, whichbecome inactivated during differentiation. Non-limiting exemplaryepigenetic modifying compound include a DNA methylation inhibitor, ahistone acetyltransferase inhibitor, a histone deacetylase inhibitor, ahistone methyltransferase inhibitor, a bromodomain inhibitor, or anycombination thereof.

In an embodiment, the histone methyltransferase inhibitor is aninhibitor of enhancer of zeste homolog 2 (EZH2). EZH2 is ahistone-lysine N-methyltransferase enzyme. Non-limiting examples of anEZH2 inhibitor that can be used in the methods provided herein include3-deazaneplanocin A (DZNep), EPZ6438, EPZ005687 (an S-adenosylmethionine(SAM) competitive inhibitor), EI1, GSK126, and UNC1999. DZNep caninhibit the hydrolysis of S-adenosyl-L-homocysteine (SAH), which is aproduct-based inhibitor of all protein methyltransferases, leading toincreased cellular concentrations of SAH which in turn inhibits EZH2.DZNep may not be specific to EZH2 and can also inhibit other DNAmethyltransferases. GSK126 is a SAM-competitive EZH2 inhibitor that has150-fold selectivity over EZH1. UNC1999 is an analogue of GSK126, and itis less selective than its counterpart GSK126.

In an embodiment, the histone methyltransferase inhibitor is DZNep. Inan embodiment, the HDAC inhibitor is a class I HDAC inhibitor, a classII HDAC inhibitor, or a combination thereof. In an embodiment, the HDACinhibitor is KD5170 (mercaptoketone-based HDAC inhibitor), MC1568 (classIIa HDAC inhibitor), TMP195 (class IIa HDAC inhibitor), or anycombination thereof. In some embodiments, HDAC inhibitor is vorinostat,romidepsin (Istodax), chidamide, panobinostat (farydak), belinostat(PXD101), panobinostat (LBH589), valproic acid, mocetinostat (MGCD0103),abexinostat (PCI-24781), entinostat (MS-275), SB939, resminostat(4SC-201), givinostat (ITF2357), quisinostat (JNJ-26481585), HBI-8000,(a benzamide HDI), kevetrin, CUDC-101, AR-42, CHR-2845, CHR-3996,4SC-202, CG200745, ACY-1215, ME-344, sulforaphane, or any variantthereof.

III. Methods of Generating Pancreatic Progenitor Cells

In aspects, the present disclosure relates to compositions and methodsof differentiating a primitive gut tube cell into a Pdx1-positivepancreatic β cell. In some cases, the method comprises contacting theprimitive gut tube cell with a composition comprising a bonemorphogenetic protein (BMP) signaling pathway inhibitor and a growthfactor from transformation growth factor β (TGF-β) superfamily. In somecases, the composition further comprises one or more additionaldifferentiation factors, which include, but not limited to, a growthfactor from fibroblast growth factor (FGF) family, a Sonic Hedgehog(SHH) pathway inhibitor, a retinoic acid (RA) signaling pathwayactivator, a protein kinase C (PKC) activator, and a Rho-associatedprotein kinase (ROCK) inhibitor.

In some cases, a method provided herein comprises generating apopulation of cells or cell cluster that comprises a Pdx1-positivepancreatic progenitor cell by contacting a population of cellscomprising a primitive gut tube cell with a first composition comprisinga BMP signaling pathway inhibitor and a growth factor from TGF-βsuperfamily, wherein the primitive gut tube cell is differentiated inthe Pdx1-positive, NKX6.1-positive pancreatic progenitor cell. In somecases, the contacting takes place for about 1 day, 2 days, or 3 days. Insome cases, the contacting takes place about 1 day. In some cases, theprimitive gut tube cell is differentiated into a Pdx1-positive,NKX6.1-negative pancreatic progenitor cell by contacting with acomposition comprising BMP signaling pathway inhibitor and a growthfactor from TGF-β superfamily. In some cases, the generating stepfurther comprises differentiating the Pdx1-positive, NKX6.1-negativepancreatic progenitor cell into a Pdx1-positive, NKX6.1-positivepancreatic progenitor cell by contacting the Pdx1-positive,NKX6.1-negative pancreatic progenitor cell with a second compositioncomprising one or more differentiation factors, which include, but notlimited to, a growth factor from TGF-β superfamily, a growth factor fromFGF family, a SHH pathway inhibitor, a RA signaling pathway activator,and a ROCK inhibitor. In some cases, the second composition does notcomprise BMP signaling pathway inhibitor.

In some cases, the method provided herein can obtain a population ofcells or cell cluster that comprises at most about 30%, at most about25%, at most about 20%, at most about 15%, at most about 10%, at mostabout 5%, at most about 4%, at most about 3%, at most about 2%, or atmost about 1% CHGA-positive cells by differentiating a population ofcells comprising primitive gut tube cells into a population of cells orcell cluster comprising Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells. In some cases, the method provided herein can obtain apopulation of cells or cell cluster that comprises at most about at mostabout 25%, at most about 20%, at most about 15%, or at most about 10%CDX2-positive cells as measured by flow cytometry by differentiating apopulation of cells comprising primitive gut tube cells into apopulation of cells or cell cluster comprising Pdx1-positive,NKX6.1-positive pancreatic progenitor cells. In some cases, the methodprovided herein can obtain a population of cells or cell cluster thatcomprises at most about 30% CHGA-positive cells and at most about 30%CDX2-positive cells by differentiating a population of cells comprisingprimitive gut tube cells into a population of cells or cell clustercomprising Pdx1-positive, NKX6.1-positive pancreatic progenitor cells.In some cases, the method provided herein can obtain a population ofcells or cell cluster that comprises at most about 20% CHGA-positivecells and at most about 5% CDX2-positive cells by differentiating apopulation of cells comprising primitive gut tube cells into apopulation of cells or cell cluster comprising Pdx1-positive,NKX6.1-positive pancreatic progenitor cells. In some cases, the methodprovided herein can obtain a population of cells or cell cluster thatcomprises at most about 15% CHGA-positive cells and at most about 3%CDX2-positive cells by differentiating a population of cells comprisingprimitive gut tube cells into a population of cells or cell clustercomprising Pdx1-positive, NKX6.1-positive pancreatic progenitor cells.

In some cases, the BMP signaling pathway inhibitor provided hereincomprises DMH-1, derivative, analogue, or variant thereof. In someembodiments, the BMP signaling pathway provided herein comprises DMH-1.In some embodiments, the method comprises contacting primitive gut tubecell with about 0.01 μM to about 10 μM, about 0.05 μM to about 5 μM,about 0.1 μM to about 1 μM, or about 0.15 μM to about 0.5 μM DMH-1. Insome embodiments, the method comprises contacting primitive gut tubecell with about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21,0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33,0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.42, 0.45, 0.48, 0.50, 0.55,0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1, 1.2, 1.4, 1.5, 1.6,1.7, 1.8, 2.0, 4.0, 6.0, 8.0, or 10 μM. In some embodiments, the methodcomprises contacting primitive gut tube cell with about 0.25 μM. In somecases, the BMP signaling pathway inhibitor as used in differentiatingthe primitive gut tube cell does not comprise LDN193189 (also named“LDN” herein).

In some cases, the methods provided herein comprise generating apopulation of cells or cell cluster that comprise a Pdx1-positive,NKX6.1-positive pancreatic progenitor cell by contacting a population ofcells comprising a primitive gut tube cell with DMH-1, or derivative,analogue, or variant thereof.

Without wishing to be bound to a particular theory, in some embodimentsof the methods disclosed herein, during differentiation of a primitivegut tube cell to a Pdx1-positive pancreatic progenitor cell, inhibitionof BMP signaling pathway can contribute to reduction in generation ofoff-target cells, for instance, cells of intestine lineage or cellspositive for CDX2 gene expression. On the other hand, in some cases,during differentiation of a primitive gut tube cell to a Pdx1-positivepancreatic progenitor cell or Pdx1-positive, NKX6.1-positive pancreaticprogenitor cell, activation of Type II receptor-mediated TGF-β signalingpathway can contribute to reduction of early induction of neurogenin 3(Ngn3) or chromogranin A (CHGA), which can, in some cases, lead togeneration of polyhormonal cells rather than mature SC-β cells, which,in some cases, are monohormonal, e.g., secreting only insulin, but notother pancreatic hormones like somatostatin or glucagon. There can becross-talk between BMP signaling pathway and TGF-β signaling pathway. Insome cases, an inhibitor of BMP signaling pathway can have side effect,for instance, blockage of, among others, Type II receptor-mediated TGF-βsignaling pathway. The inhibition of Type II receptor-mediated TGF-βsignaling pathway, as illustrated in FIG. 15 , for instance by arelatively less selective BMP signaling pathway inhibitor, LDN193189,can lead to early NGN3/CHGA induction, thereby generation ofpolyhormonal cells. Without wishing to be bound by a certain theory, insome cases, use of a highly selective BMP signaling pathway inhibitor,for instance, DMH-1 or its derivate, analogue, or variant, can have lessinhibitory effect on Type II receptor-mediated TGF-β signaling pathway.In some other cases, without wishing to be bound to a particular theory,co-incubation with a growth factor from TGF-β superfamily together witha BMP signaling pathway inhibitor can result in selective inhibition ofBMP signaling pathway, while maintaining relatively high activationlevel of Type II receptor-mediated TGF-β signaling pathway. In somecases, co-incubation with a growth factor from TGF-β superfamilytogether with a BMP signaling pathway inhibitor result in reducedgeneration of off-target cells, e.g., CDX2-positive cells, as well asreduced generation of polyhormonal cells, for instance, as a result ofearly induction of NGN3 or CHGA in the cells differentiated from theprimitive gut tube cells.

In some aspects, the present disclosure provides a method of producing aNKX6 positive pancreatic progenitor cell from a Pdx1-positive pancreaticprogenitor cell comprising contacting a population of cells comprisingPdx1-positive pancreatic progenitor cells or precursors under conditionsthat promote cell clustering with at least two β cell-maturation factorscomprising a) at least one growth factor from the fibroblast growthfactor (FGF) family, b) a sonic hedgehog pathway inhibitor, andoptionally c) a low concentration of a retinoic acid (RA) signalingpathway activator, for a period of at least five days to induce thedifferentiation of at least one Pdx1-positive pancreatic progenitor cellin the population into NKX6-1-positive pancreatic progenitor cells,wherein the NKX6-1-positive pancreatic progenitor cells express NKX6-1.

In some embodiments, at least 10% of the Pdx1-positive pancreaticprogenitor cells in the population are induced to differentiate intoNKX6-1-positive pancreatic progenitor cells. In some embodiments, atleast 95% of the Pdx1-positive pancreatic progenitor cells in thepopulation are induced to differentiate into NKX6-1-positive pancreaticprogenitor cells. In some embodiments, the NKX6-1-positive pancreaticprogenitor cells express Pdx1, NKX6-1, and FoxA2. In some embodiments,the Pdx1-positive pancreatic progenitor cells are produced from apopulation of pluripotent stem cells selected from the group consistingof embryonic stem cells and induced pluripotent stem cells.

IV. Stem Cells and Reprogramming

Provided herein is use of stem cells for producing SC-β cells (e.g.,mature pancreatic β cells or β-like cells) or precursors thereof. In anembodiment, germ cells may be used in place of, or with, the stem cellsto provide at least one SC-β cell, using similar protocols as describedin U.S. Patent Application Publication No. US20150240212 andUS20150218522, each of which is herein incorporated by reference in itsentirety. Suitable germ cells can be prepared, for example, fromprimordial germ cells present in human fetal material taken about 8-11weeks after the last menstrual period. Illustrative germ cellpreparation methods are described, for example, in Shamblott et al.,Proc. Natl. Acad. Sci. USA 95:13726, 1998 and U.S. Pat. No. 6,090,622.

Provided herein are compositions and methods of generating SC-β cells(e.g., pancreatic β cells). Generally, the at least one SC-β cell orprecursor thereof, e.g., pancreatic progenitors produced according tothe methods disclosed herein can comprise a mixture or combination ofdifferent cells, e.g., for example a mixture of cells such as primitivegut tube cells, Pdx1-positive pancreatic progenitors, Pdx1-positive,NKX6-1-positive pancreatic progenitors, Ngn3-positive endocrineprogenitor cells, insulin-positive endocrine cell (e.g., β-like cells),and/or other pluripotent or stem cells.

The at least one SC-β cell or precursor thereof can be producedaccording to any suitable culturing protocol to differentiate a stemcell or pluripotent cell to a desired stage of differentiation. In someembodiments, the at least one SC-β cell or the precursor thereof areproduced by culturing at least one pluripotent cell for a period of timeand under conditions suitable for the at least one pluripotent cell todifferentiate into the at least one SC-β cell or the precursor thereof.

In some embodiments, the at least one SC-β cell or precursor thereof isa substantially pure population of SC-β cells or precursors thereof. Insome embodiments, a population of SC-β cells or precursors thereofcomprises a mixture of pluripotent cells or differentiated cells. Insome embodiments, a population SC-β cells or precursors thereof aresubstantially free or devoid of embryonic stem cells or pluripotentcells or iPS cells.

In some embodiments, a somatic cell, e.g., fibroblast can be isolatedfrom a subject, for example as a tissue biopsy, such as, for example, askin biopsy, and reprogrammed into an induced pluripotent stem cell forfurther differentiation to produce the at least one SC-β cell orprecursor thereof for use in the compositions and methods describedherein. In some embodiments, a somatic cell, e.g., fibroblast ismaintained in culture by methods known by one of ordinary skill in theart, and in some embodiments, propagated prior to being converted intoSC-β cells by the methods as disclosed herein.

In some embodiments, the at least one SC-β cell or precursor thereof aremaintained in culture by methods known by one of ordinary skills in theart, and in some embodiments, propagated prior to being converted intoSC-β cells by the methods as disclosed herein.

Further, at least one SC-β cell or precursor thereof, e.g., pancreaticprogenitor can be from any mammalian species, with non-limiting examplesincluding a murine, bovine, simian, porcine, equine, ovine, or humancell. For clarity and simplicity, the description of the methods hereinrefers to a mammalian at least one SC-β cell or precursor thereof but itshould be understood that all of the methods described herein can bereadily applied to other cell types of at least one SC-β cell orprecursor thereof. In some embodiments, the at least one SC-β cell orprecursor thereof is derived from a human individual.

Stem Cells

Embodiments of the present disclosure can related to use of stem cellsfor generation of pancreatic β cells or precursors thereof. The term“stem cell” as used herein can refer to a cell (e.g., plant stem cell,vertebrate stem cell) that has the ability both to self-renew and togenerate a differentiated cell type (Morrison et al., (1997) Cell88:287-298). In the context of cell ontogeny, the adjective“differentiated”, or “differentiating” is a relative term. A“differentiated cell” can be a cell that has progressed further down thedevelopmental pathway than the cell it is being compared with. Thus,pluripotent stem cells can differentiate into lineage-restrictedprogenitor cells (e.g., mesodermal stem cells), which in turn candifferentiate into cells that are further restricted (e.g., neuronprogenitors), which can differentiate into end-stage cells (e.g.,terminally differentiated cells, e.g., neurons, cardiomyocytes, etc.),which play a characteristic role in a certain tissue type, and can orcannot retain the capacity to proliferate further. Stem cells can becharacterized by both the presence of specific markers (e.g., proteins,RNAs, etc.) and the absence of specific markers. Stem cells can also beidentified by functional assays both in vitro and in vivo, particularlyassays relating to the ability of stem cells to give rise to multipledifferentiated progeny. In an embodiment, the host cell is an adult stemcell, a somatic stem cell, a non-embryonic stem cell, an embryonic stemcell, hematopoietic stem cell, an include pluripotent stem cells, and atrophoblast stem cell.

Stem cells of interest, e.g., that can be used in the method providedherein, can include pluripotent stem cells (PSCs). The term “pluripotentstem cell” or “PSC” as used herein can refer to a stem cell capable ofproducing all cell types of the organism. Therefore, a PSC can give riseto cells of all germ layers of the organism (e.g., the endoderm,mesoderm, and ectoderm of a vertebrate). Pluripotent cells can becapable of forming teratomas and of contributing to ectoderm, mesoderm,or endoderm tissues in a living organism. Pluripotent stem cells ofplants can be capable of giving rise to all cell types of the plant(e.g., cells of the root, stem, leaves, etc.).

Embodiments of the present disclosure can related to use of PSCs forgeneration of pancreatic β cells or precursors thereof. PSCs of animalscan be derived in a number of different ways. For example, embryonicstem cells (ESCs) can be derived from the inner cell mass of an embryo(Thomson et. al, Science. 1998 Nov. 6; 282(5391):1145-7) whereas inducedpluripotent stem cells (iPSCs) can be derived from somatic cells(Takahashi et. al, Cell. 2007 Nov. 30; 131(5):861-72; Takahashi et. al,Nat Protoc. 2007; 2(12):3081-9; Yu et. al, Science. 2007 Dec. 21;318(5858):1917-20. Epub 2007 Nov. 20). Because the term PSC can refer topluripotent stem cells regardless of their derivation, the term PSC canencompass the terms ESC and iPSC, as well as the term embryonic germstem cells (EGSC), which are another example of a PSC. PSCs can be inthe form of an established cell line, they can be obtained directly fromprimary embryonic tissue, or they can be derived from a somatic cell.

Embodiments of the present disclosure can related to use of ESCs forgeneration of pancreatic β cells or precursors thereof. By “embryonicstem cell” (ESC) can be meant a PSC that is isolated from an embryo,typically from the inner cell mass of the blastocyst. ESC lines arelisted in the NIH Human Embryonic Stem Cell Registry, e.g. hESBGN-01,hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3,HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMediHospital-Seoul National University); HSF-1, HSF-6 (University ofCalifornia at San Francisco); and H1, H7, H9, H13, H14 (Wisconsin AlumniResearch Foundation (WiCell Research Institute)). Stem cells of interestalso include embryonic stem cells from other primates, such as Rhesusstem cells and marmoset stem cells. The stem cells can be obtained fromany mammalian species, e.g. human, equine, bovine, porcine, canine,feline, rodent, e.g. mice, rats, hamster, primate, etc. (Thomson et al.(1998) Science 282:1145; Thomson et al. (1995) Proc. Natl. Acad. Sci USA92:7844; Thomson et al. (1996) Biol. Reprod. 55:254; Shamblott et al.,Proc. Natl. Acad. Sci. USA 95:13726, 1998). In culture, ESCs can grow asflat colonies with large nucleo-cytoplasmic ratios, defined borders andprominent nucleoli. In addition, ESCs can express SSEA-3, SSEA-4,TRA-1-60, TRA-1-81, and Alkaline Phosphatase, but not SSEA-1. Examplesof methods of generating and characterizing ESCs can be found in, forexample, U.S. Pat. Nos. 7,029,913, 5,843,780, and 6,200,806, each ofwhich is incorporated herein by its entirety. Methods for proliferatinghESCs in the undifferentiated form are described in WO 99/20741, WO01/51616, and WO 03/020920, each of which is incorporated herein by itsentirety.

By “embryonic germ stem cell” (EGSC) or “embryonic germ cell” or “EGcell”, it can be meant a PSC that is derived from germ cells and/or germcell progenitors, e.g. primordial germ cells, e.g. those that can becomesperm and eggs. Embryonic germ cells (EG cells) are thought to haveproperties similar to embryonic stem cells as described above. Examplesof methods of generating and characterizing EG cells may be found in,for example, U.S. Pat. No. 7,153,684; Matsui, Y., et al., (1992) Cell70:841; Shamblott, M., et al. (2001) Proc. Natl. Acad. Sci. USA 98: 113;Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726; andKoshimizu, U., et al. (1996) Development, 122:1235, each of which areincorporated herein by its entirety.

Embodiments of the present disclosure can related to use of iPSCs forgeneration of pancreatic β cells or precursors thereof. By “inducedpluripotent stem cell” or “iPSC”, it can be meant a PSC that is derivedfrom a cell that is not a PSC (e.g., from a cell this is differentiatedrelative to a PSC). iPSCs can be derived from multiple different celltypes, including terminally differentiated cells. iPSCs can have an EScell-like morphology, growing as flat colonies with largenucleo-cytoplasmic ratios, defined borders and prominent nuclei. Inaddition, iPSCs can express one or more key pluripotency markers knownby one of ordinary skill in the art, including but not limited toAlkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181,TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. Examples ofmethods of generating and characterizing iPSCs can be found in, forexample, U.S. Patent Publication Nos. US20090047263, US20090068742,US20090191159, US20090227032, US20090246875, and US20090304646, each ofwhich are incorporated herein by its entirety. Generally, to generateiPSCs, somatic cells are provided with reprogramming factors (e.g. Oct4,SOX2, KLF4, MYC, Nanog, Lin28, etc.) known in the art to reprogram thesomatic cells to become pluripotent stem cells.

Embodiments of the present disclosure can related to use of somaticcells for generation of pancreatic β cells or precursors thereof. By“somatic cell”, it can be meant any cell in an organism that, in theabsence of experimental manipulation, does not ordinarily give rise toall types of cells in an organism. In other words, somatic cells can becells that have differentiated sufficiently that they may not naturallygenerate cells of all three germ layers of the body, e.g. ectoderm,mesoderm and endoderm. For example, somatic cells can include bothneurons and neural progenitors, the latter of which is able to naturallygive rise to all or some cell types of the central nervous system butcannot give rise to cells of the mesoderm or endoderm lineages

In certain examples, the stem cells can be undifferentiated (e.g. a cellnot committed to a specific lineage) prior to exposure to at least onedifferentiation factor or composition according to the methods asdisclosed herein, whereas in other examples it can be desirable todifferentiate the stem cells to one or more intermediate cell typesprior to exposure of the at least one differentiation factor orcomposition described herein. For example, the stems cells can displaymorphological, biological or physical characteristics ofundifferentiated cells that can be used to distinguish them fromdifferentiated cells of embryo or adult origin. In some examples,undifferentiated cells can appear in the two dimensions of a microscopicview in colonies of cells with high nuclear/cytoplasmic ratios andprominent nucleoli. The stem cells can be themselves (for example,without substantially any undifferentiated cells being present) or canbe used in the presence of differentiated cells. In certain examples,the stem cells can be cultured in the presence of) suitable nutrientsand optionally other cells such that the stem cells can grow andoptionally differentiate. For example, embryonic fibroblasts orfibroblast-like cells can be present in the culture to assist in thegrowth of the stem cells. The fibroblast can be present during one stageof stem cell growth but not necessarily at all stages. For example, thefibroblast can be added to stem cell cultures in a first culturing stageand not added to the stem cell cultures in one or more subsequentculturing stages.

Stem cells used in all aspects of the present invention can be any cellsderived from any kind of tissue (for example embryonic tissue such asfetal or pre-fetal tissue, or adult tissue), which stem cells can havethe characteristic of being capable under appropriate conditions ofproducing progeny of different cell types, e.g. derivatives of all of atleast one of the 3 germinal layers (endoderm, mesoderm, and ectoderm).These cell types can be provided in the form of an established cellline, or they can be obtained directly from primary embryonic tissue andused immediately for differentiation. Included are cells listed in theNIH Human Embryonic Stem Cell Registry, e.g. hESBGN-01, hESBGN-02,hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4,HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-SeoulNational University); HSF-1, FISF-6 (University of California at SanFrancisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni ResearchFoundation (WiCell Research Institute)). In some embodiments, the sourceof human stem cells or pluripotent stem cells used forchemically-induced differentiation into mature, insulin positive cellsdid not involve destroying a human embryo. In some embodiments, thesource of human stem cells or pluripotent stem cells used forchemically-induced differentiation into mature, insulin positive cellsdo not involve destroying a human embryo.

In another example, the stem cells can be isolated from tissue includingsolid tissue. In some embodiments, the tissue is skin, fat tissue (e.g.adipose tissue), muscle tissue, heart or cardiac tissue. In otherembodiments, the tissue is for example but not limited to, umbilicalcord blood, placenta, bone marrow, or chondral.

Stem cells that can be used in the methods provided herein can alsoinclude embryonic cells of various types, exemplified by human embryonicstem (hES) cells, as described by Thomson et al, (1998) Science282:1145; embryonic stem cells from other primates, such as Rhesus stemcells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844);marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254); andhuman embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad.Sci. USA 95:13726, 1998). Also applicable to the methods provided hereincan be lineage committed stem cells, such as mesodermal stem cells andother early cardiogenic cells (see Reyes et al, (2001) Blood98:2615-2625; Eisenberg & Bader (1996) Circ Res. 78(2):205-16; etc.) Thestem cells can be obtained from any mammalian species, e.g. human,equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats,hamster, primate, etc. In some embodiments, a human embryo was notdestroyed for the source of pluripotent cell used on the methods andcompositions as disclosed herein. In some embodiments, a human embryo isnot destroyed for the source of pluripotent cell used on the methods andcompositions as disclosed herein.

A mixture of cells from a suitable source of endothelial, muscle, and/orneural stem cells can be harvested from a mammalian donor for thepurpose of the present disclosure. A suitable source is thehematopoietic microenvironment. For example, circulating peripheralblood, preferably mobilized (e.g., recruited), may be removed from asubject. In an embodiment, the stem cells can be reprogrammed stemcells, such as stem cells derived from somatic or differentiated cells.In such an embodiment, the de-differentiated stem cells can be forexample, but not limited to, neoplastic cells, tumor cells and cancercells or alternatively induced reprogrammed cells such as inducedpluripotent stem cells or iPS cells.

In some embodiments, the pancreatic β cell as described herein can bederived from one or more of trichocytes, keratinocytes, gonadotropes,corticotropes, thyrotropes, somatotropes, lactotrophs, chromaffin cells,parafollicular cells, glomus cells melanocytes, nevus cells, Merkelcells, odontoblasts, cementoblasts corneal keratocytes, retina Mullercells, retinal pigment epithelium cells, neurons, glias (e.g.,oligodendrocyte astrocytes), ependymocytes, pinealocytes, pneumocytes(e.g., type I pneumocytes, and type II pneumocytes), clara cells, gobletcells, G cells, D cells, ECL cells, gastric chief cells, parietal cells,foveolar cells, K cells, D cells, I cells, goblet cells, paneth cells,enterocytes, microfold cells, hepatocytes, hepatic stellate cells (e.g.,Kupffer cells from mesoderm), cholecystocytes, centroacinar cells,pancreatic stellate cells, pancreatic α cells, pancreatic β cells,pancreatic δ cells, pancreatic F cells (e.g., PP cells), pancreatic ccells, thyroid (e.g., follicular cells), parathyroid (e.g., parathyroidchief cells), oxyphil cells, urothelial cells, osteoblasts, osteocytes,chondroblasts, chondrocytes, fibroblasts, fibrocytes, myoblasts,myocytes, myosatellite cells, tendon cells, cardiac muscle cells,lipoblasts, adipocytes, interstitial cells of cajal, angioblasts,endothelial cells, mesangial cells (e.g., intraglomerular mesangialcells and extraglomerular mesangial cells), juxtaglomerular cells,macula densa cells, stromal cells, interstitial cells, telocytes simpleepithelial cells, podocytes, kidney proximal tubule brush border cells,sertoli cells, leydig cells, granulosa cells, peg cells, germ cells,spermatozoon ovums, lymphocytes, myeloid cells, endothelial progenitorcells, endothelial stem cells, angioblasts, mesoangioblasts, pericytemural cells, splenocytes (e.g., T lymphocytes, B lymphocytes, dendriticcells, microphages, leukocytes), trophoblast stem cells, or anycombination thereof.

Reprogramming

The term “reprogramming” as used herein can refer to the process thatalters or reverses the differentiation state of a somatic cell. The cellcan either be partially or terminally differentiated prior to thereprogramming. Reprogramming can encompass complete reversion of thedifferentiation state of a somatic cell to a pluripotent cell. Suchcomplete reversal of differentiation can produce an induced pluripotent(iPS) cell. Reprogramming as used herein can also encompass partialreversion of a cells differentiation state, for example to a multipotentstate or to a somatic cell that is neither pluripotent or multipotent,but is a cell that has lost one or more specific characteristics of thedifferentiated cell from which it arises, e.g. direct reprogramming of adifferentiated cell to a different somatic cell type. Reprogramming caninvolve alteration, e.g., reversal, of at least some of the heritablepatterns of nucleic acid modification (e.g., methylation), chromatincondensation, epigenetic changes, genomic imprinting, etc., that occurduring cellular differentiation as a zygote develops into an adult.

As used herein, the term “reprogramming factor” can refer to a moleculethat is associated with cell “reprogramming,” that is, differentiation,and/or de-differentiation, and/or transdifferentiation, such that a cellconverts to a different cell type or phenotype. Reprogramming factorsgenerally affect expression of genes associated with celldifferentiation, de-differentiation and/or transdifferentiation.Transcription factors are examples of reprogramming factors.

The term “differentiation” and their grammatical equivalents as usedherein can refer to the process by which a less specialized cell (e.g.,a more naive cell with a higher cell potency) becomes a more specializedcell type (e.g., a less naive cell with a lower cell potency); and thatthe term “de-differentiation” can refer to the process by which a morespecialized cell becomes a less specialized cell type (e.g., a morenaive cell with a higher cell potency); and that the term“transdifferentiation” refers to the process by which a cell of aparticular cell type converts to another cell type without significantlychanging its “cell potency” or “naivety” level. Without wishing to bebound by theory, it is thought that cells “transdifferentiate” when theyconvert from one lineage-committed cell type or terminallydifferentiated cell type to another lineage-committed cell type orterminally differentiated cell type, without significantly changingtheir “cell potency” or “naivety” level.

As used herein, the term “cell potency” is to be understood as referringto the ability of a cell to differentiate into cells of differentlineages. For example, a pluripotent cell (e.g., a stem cell) has thepotential to differentiate into cells of any of the three germ layers,that is, endoderm (interior stomach lining, gastrointestinal tract, thelungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm(epidermal tissues and nervous system), and accordingly has high cellpotency; a multipotent cell (e.g., a stem cell or an induced stem cellof a certain type) has the ability to give rise to cells from amultiple, but limited, number of lineages (such as hematopoietic stemcells, cardiac stem cells, or neural stem cells, etc) comparatively hasa lower cell potency than pluripotent cells. Cells that are committed toa particular lineage or are terminally differentiated can have yet alower cell potency. Specific examples of transdifferentiation known inthe art include the conversion of e.g., fibroblasts beta cells or frompancreatic exocrine cells to beta cells etc.

Accordingly, the cell may be caused to differentiate into a more naivecell (e.g., a terminally differentiated cell may be differentiated to bemultipotent or pluripotent); or the cell may be caused tode-differentiate into a less naive cell (e.g., a multipotent orpluripotent cell can be differentiated into a lineage-committed cell ora terminally differentiated cell). However, in an embodiment, the cellmay be caused to convert or transdifferentiate from one cell type (orphenotype) to another cell type (or phenotype), for example, with asimilar cell potency level. Accordingly, in an embodiment of the presentdisclosure, the inducing steps of the present disclosure can reprogramthe cells of the present disclosure to differentiate, de-differentiateand/or transdifferentiate. In an embodiment of the present disclosure,the inducing steps of the present disclosure may reprogram the cells totransdifferentiate.

Methods of reprogramming or inducing a particular type of cell to becomeanother type of cell, for example, by differentiation,de-differentiation and/or transdifferentiation using one or moreexogenous polynucleotide or polypeptide reprogramming factors are knownto the person skilled in the art. Such methods may rely on theintroduction of genetic material encoding one or more transcriptionfactor(s) or other polypeptide(s) associated with cell reprogramming.For example, Pdx1, Ngn3 and MafA, or functional fragments thereof areall known to encode peptides that can induce cell differentiation,de-differentiation and/or transdifferentiation of the cells of thepresent disclosure. In some methods known to the person skilled in theart, exogenous polypeptides (e.g. recombinant polypeptides) encoded byreprogramming genes (such as the above genes) are contacted with thecells to induce, for example, cells of the present disclosure. Theperson skilled in the art will appreciate that other genes may beassociated with reprogramming of cells, and exogenous molecules encodingsuch genes (or functional fragments thereof) and the encodedpolypeptides are also considered to be polynucleotide or polypeptidereprogramming factors (e.g. polynucleotides or polypeptides that in turnaffect expression levels of another gene associated with cellreprogramming). For example, it has been shown that the introduction ofexogenous polynucleotide or polypeptide epigenetic gene silencers thatdecrease p53 inactivation increase the efficiency of inducing inducedpluripotent stem cells (iPSC). Accordingly, exogenous polynucleotides orpolypeptides encoding epigenetic silencers and other genes or proteinsthat may be directly or indirectly involved in cell reprogramming orincreasing cell programming efficiency would be considered to constitutean exogenous polynucleotide or polypeptide reprogramming factor. Theperson skilled in the art will appreciate that other methods ofinfluencing cell reprogramming exist, such as introducing RNAi molecules(or genetic material encoding RNAi molecules) that can knock downexpression of genes involved in inhibiting cell reprogramming.Accordingly, any exogenous polynucleotide molecule or polypeptidemolecule that is associated with cell reprogramming, or enhances cellreprogramming, is to be understood to be an exogenous polynucleotide orpolypeptide reprogramming factor as described herein.

In some embodiments of the present disclosure, the method excludes theuse of reprogramming factor(s) that are not small molecules. However, itwill be appreciated that the method can utilize “routine” tissue culturecomponents such as culture media, serum, serum substitutes, supplements,antibiotics, etc, such as RPMI, Renal Epithelial Basal Medium (REBM),Dulbecco's Modified Eagle Medium (DMEM), MCDB131 medium, CMRL 1066medium, F12, foetal calf serum (FCS), foetal bovine serum (FBS), bovineserum albumin (BSA), D-glucose, L-glutamine, GlutaMAX™-1 (dipeptide,L-alanine-L-glutamine), B27, heparin, progesterone, putrescine, laminin,nicotinamide, insulin, transferrin, sodium selenite, selenium,ethanolamine, human epidermal growth factor (hEGF), basic fibroblastgrowth factor (bFGF), hydrocortisone, epinephrine, normacin, penicillin,streptomycin, gentamicin and amphotericin, etc. It is to be understoodthat these typical tissue culture components (and other similar tissueculture components that are routinely used in tissue culture) are notsmall molecule reprogramming molecules for the purposes of the presentdisclosure. These components are either not small molecules as definedherein and/or are not reprogramming factors as defined herein.

Accordingly, in an embodiment, the present disclosure does not involve aculturing step of the cell(s) with one or more exogenous polynucleotideor polypeptide reprogramming factor(s). Accordingly, in an embodiment,the method of the present disclosure does not involve the introductionof one or more exogenous polynucleotide or polypeptide reprogrammingfactor(s), e.g., by introducing transposons, viral transgenic vectors(such as retroviral vectors), plasmids, mRNA, miRNA, peptides, orfragments of any of these molecules, that are involved in producinginduced β cells or, otherwise, inducing cells of the present disclosureto differentiate, de-differentiation and/or transdifferentiate.

That is, in an embodiment, the method occurs in the absence of one ormore exogenous polynucleotide or polypeptide reprogramming factor(s).Accordingly, it is to be understood that in an embodiment, the method ofthe present disclosure utilizes small molecules (e.g., HDAC inhibitors)to reprogram cells, without the addition of polypeptide transcriptionfactors; other polypeptide factors specifically associated with inducingdifferentiation, de-differentiation, and/or transdifferentiation;polynucleotide sequences encoding polypeptide transcription factors,polynucleotide sequences encoding other polypeptide factors specificallyassociated with inducing differentiation, de-differentiation, and/ortransdifferentiation; mRNA; interference RNA; microRNA and fragmentsthereof.

V. Xeno-Free Culture Medium

In aspects, the present disclosure relates to a method of generatingpancreatic β cells, e.g., SC-β cells, which comprises differentiatingprogenitor cells (e.g., stem cells like iPSC cells, definitive endodermcells, primitive gut tube cells, Pdx1-positive pancreatic progenitorcells, NKX6.1-positive pancreatic progenitor cells, or insulin-positiveendocrine cells) in a xeno-free culture medium. A xeno-free medium forculturing cells and/or cell clusters of originated from an animal canhave no product from other animals. In some cases, a xeno-free mediumfor culturing human cells and/or cell clusters can have no products fromany non-human animals. For example, a xeno-free medium for culturinghuman cells and/or cell clusters can comprise human serum albumin (HSA)or human platelet lysate (PLT) instead of fetal bovine serum (FBS) orbovine serum albumin (BSA).

In some embodiments, a method provided herein comprises generatingpancreatic β cells, e.g., SC-β cells, by differentiating progenitorcells (e.g., stem cells like iPSC cells, definitive endoderm cells,primitive gut tube cells, Pdx1-positive pancreatic progenitor cells,NKX6.1-positive pancreatic progenitor cells, or insulin-positiveendocrine cells) in a culture medium lacking serum albumin. In somecases, a population of cells or cell cluster comprising pancreatic βcells generated by a method provided herein that does not use serumalbumin or uses HSA in the culture medium can have significantimprovement as compared to a population of cells or cell clustercomprising pancreatic β cells generated by an otherwise identical methodbut using BSA instead. The improvement can include higher percentage ofpancreatic β cells in the final cell population obtained, higher GSISresponses (e.g., more insulin release in response to glucose challenge),higher GSIS stimulation index, more homogeneity of distribution ofpancreatic β cells in the cell cluster generated, or any combinationthereof.

In some embodiments, a method provided herein comprises differentiatinga population of cells comprising a stem cell, e.g., a hES cell or iPScell, in a culture medium comprising human serum albumin (HSA). In somecases, the stem cell is differentiated into a definitive endoderm cell.In some embodiments, a method provided herein comprises differentiatinga population of cells comprising a definitive endoderm cell in a culturemedium comprising human serum albumin (HSA). In some cases, thedefinitive endoderm cell is differentiated into a primitive gut tubecell. In some embodiments, a method provided herein comprisesdifferentiating a population of cells comprising a primitive gut tubecell in a culture medium comprising human serum albumin (HSA). In somecases, the primitive gut tube cell is differentiated into aPdx1-positive pancreatic progenitor cell (e.g., Pdx1-positive,NKX6.1-negative pancreatic progenitor cell or Pdx1-positive,NKX6.1-positive pancreatic progenitor cell). In some embodiments, amethod provided herein comprises differentiating a population of cellscomprising a Pdx1-positive, NKX6.1-negative pancreatic progenitor cellin a culture medium comprising human serum albumin (HSA). In some case,the Pdx1-positive, NKX6.1-negative pancreatic progenitor cell isdifferentiated into a Pdx1-positive, NKX6.1-positive pancreaticprogenitor cell. In some embodiments, a method provided herein comprisesdifferentiating a population of cells comprising a Pdx1-positive,NKX6.1-positive pancreatic progenitor cell in a culture mediumcomprising human serum albumin (HSA). In some cases, the Pdx1-positive,NKX6.1-positive pancreatic progenitor cell is differentiated into aninsulin-positive endocrine cell. In some embodiments, a method providedherein comprises differentiating a population of cells comprising aninsulin-positive endocrine cell in a culture medium comprising humanserum albumin (HSA). In some cases, the insulin-positive endocrine cellis differentiated into a pancreatic β cell, e.g., SC-β cell.

In some embodiments, the methods provided herein comprise use of culturemedium comprising about 0.001% (w/v) to about 5% (w/v), about 0.005%(w/v) to about 4% (w/v), about 0.01% (w/v) to about 3% (w/v), about0.02% (w/v) to about 2.5% (w/v), about 0.03% (w/v) to about 2% (w/v),about 0.04% (w/v) to about 1% (w/v), about 0.045% (w/v) to about 0.5%(w/v), or about 0.05% (w/v) to about 0.1% (w/v) HSA. In someembodiments, the methods provided herein comprise use of culture mediumcomprising about 0.001%, 0.002%, 0.0025%, 0.005%, 0.0075%, 0.01%,0.0125%, 0.015%, 0.0175%, 0.02%, 0.0225%, 0.025%, 0.0275%, 0.03%,0.0325%, 0.035%, 0.0375%, 0.04%, 0.0425%, 0.045%, 0.0475%, 0.05%,0.0525%, 0.055%, 0.575%, 0.06%, 0.0625%, 0.065%, 0.0675%, 0.07%,0.0725%, 0.075%, 0.0775%, 0.08%, 0.085%, 0.09%, 0.1%, 0.12%, 0.15%,0.2%, 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%,2.5%, 3%, or 4%, 5% (w/v) HSA. The term “w/v” is short for percentage ofweight/volume or weight per volume. For instance, 1 mg HSA in 100 mLculture medium has a concentration of 1% (w/v).

In some cases, the method provided herein can obtain a population ofcells or cell cluster that comprises at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 86%, at least about 87%, at least about88%, at least about 89%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, or at leastabout 95% Pdx1-positive cells by differentiating a population of cellscomprising primitive gut tube cells into a population of cells or cellcluster comprising Pdx1-positive, NKX6.1-negative pancreatic progenitorcells. In some cases, the method provided herein can obtain a populationof cells or cell cluster that comprises at most about 60%, at most about50%, at most about 40%, at most about 35%, at most about 30%, at mostabout 25%, at most about 22%, at most about 20%, at most about 18%, atmost about 15%, at most about 14%, at most about 13%, at most about 11%,at most about 12%, at most about 10%, or at most about 5% CDX2-positivecells by differentiating a population of cells comprising primitive guttube cells into a population of cells or cell cluster comprisingPdx1-positive, NKX6.1-negative pancreatic progenitor cells.

VI. Method of Generating Stem Cell Derived Beta Cells

Provided herein are methods of generating SC-β cells (e.g., pancreatic βcells). The detailed protocols of generating endocrine cells the stemcells to provide at least one SC-β cell are described in U.S. PatentApplication Publication No. US20150240212 and US20150218522, each ofwhich is herein incorporated by reference in its entirety.

The endoderm can give rise to digestive and respiratory tracts, thyroid,liver, and pancreas. Representative disease of endoderm lineages is type1 diabetes resulting from destruction of the insulin-producing β cells.Generation of functional β cells from human pluripotent stem cells(hPSC) in vitro can be practical, renewable cell source for replacementcell therapy for type 1 diabetes. The embryotic stem (ES) cells that aregenerated from the inner cell mass of blastocyst-stage embryos representa promising source of cells for transplantation or cell-based therapy ofany damaged cells. They can be maintained in culture, renew forthemselves, and proliferate unlimitedly as undifferentiated ES cells.The ES cells are capable of differentiating into all cell types of thebody as the ectoderm, mesoderm, and endoderm lineage cells or tissues.The major benefit of ES cells is stable self-renewal in culture and thepotential to differentiate.

The definitive endoderm can be generated in vivo from the inner cellmass by the process of gastrulation of embryogenesis, in which epiblastcells are instructed to form the three germ layers. Definitive endodermcan give rise to diverse cells and tissues that contribute to vitalorgans as the pancreatic β cells, liver hepatocytes, lung alveolarcells, thyroid, thymus, and the epithelial lining of the alimentary andrespiratory tract. It is different from the primitive endoderm ofextraembryonic tissues, which can give rise to the visceral and parietalendoderm. The definitive endoderm derived from ES cells is theoreticallycapable of becoming any endoderm derivatives, and directing ES cellsinto the endoderm lineage is a prerequisite for generating therapeuticendoderm derivatives.

Precise patterning of anterior-posterior axis of the definitive endodermcan eventually form the primitive gut tube. The definitiveendoderm-derived primitive gut tube induces the pharynx, esophagus,stomach, duodenum, small and large intestine along theanterior-posterior axis as well as associated organs, includingpancreas, lung, thyroid, thymus, parathyroid, and liver. The anteriorportion of the foregut of the primitive gut tube becomes lung, thyroid,esophagus, and stomach. The pancreas, liver, and duodenum originate fromthe posterior portion of the foregut. The midgut and hindgut ofprimitive gut tube gives rise to the small and large intestine. Theanterior foregut expresses developmental markers, NK2 homeobox 1(NKX2-1) and SRY (sex determining region Y)-box 2 (SOX2); the posteriorforegut expresses hematopoietically expressed homeobox (HHEX),pancreatic and duodenal homeobox 1 (PDX1), one cut homeobox 1 (ONECUT1,known as HNF6), and hepatocyte nuclear factor 4 alpha (HNF4A); and themidgut/hindgut expresses caudal type homeobox 1 (CDX1), caudal typehomeobox 2 (CDX2), and motor neuron and pancreas homeobox 1 (MNX1) (3,19, 20).

The successful differentiation to pancreatic β cells should require thatdifferentiated cells synthesize and secrete physiologically appropriateamounts of insulin. An exemplary stepwise protocol directing hPSC celldifferentiation is developed, which entails differentiation processesthat recapitulates the major stages of normal pancreatic endocrinedevelopment (FIG. 5 ). The differentiation of hPSC cells tohormone-expressing pancreatic endocrine cells is conducted by transitinghPSC cells through major stages of embryonic development;differentiation to mesendoderm and definitive endoderm, establishment ofthe primitive gut endoderm, patterning of the posterior foregut, andspecification and maturation of pancreatic endoderm and endocrineprecursors. Through these stages, hPSC cells can obtain pancreaticendocrine phenotype and ability of glucose responsive insulin secretionin vitro.

Generally, the at least one SC-β cell or precursor thereof, e.g.,pancreatic progenitors produced according to the methods disclosedherein can comprise a mixture or combination of different cells, e.g.,for example a mixture of cells such as a Pdx1-positive pancreaticprogenitors, pancreatic progenitors co-expressing Pdx1 and NKX6-1, aNgn3-positive endocrine progenitor cell, an insulin-positive endocrinecell (e.g., a β-like cell), and an insulin-positive endocrine cell,and/or other pluripotent or stem cells.

The at least one SC-β cell or precursor thereof can be producedaccording to any suitable culturing protocol to differentiate a stemcell or pluripotent cell to a desired stage of differentiation. In someembodiments, the at least one SC-β cell or the precursor thereof areproduced by culturing at least one pluripotent cell for a period of timeand under conditions suitable for the at least one pluripotent cell todifferentiate into the at least one SC-β cell or the precursor thereof.

In some embodiments, the at least one SC-β cell or precursor thereof isa substantially pure population of SC-β cells or precursors thereof. Insome embodiments, a population of SC-β cells or precursors thereofcomprises a mixture of pluripotent cells or differentiated cells. Insome embodiments, a population SC-β cells or precursors thereof aresubstantially free or devoid of embryonic stem cells or pluripotentcells or iPS cells.

In some embodiments, a somatic cell, e.g., fibroblast can be isolatedfrom a subject, for example as a tissue biopsy, such as, for example, askin biopsy, and reprogrammed into an induced pluripotent stem cell forfurther differentiation to produce the at least one SC-β cell orprecursor thereof for use in the compositions and methods describedherein. In some embodiments, a somatic cell, e.g., fibroblast ismaintained in culture by methods known by one of ordinary skill in theart, and in some embodiments, propagated prior to being converted intoSC-β cells by the methods as disclosed herein.

In some embodiments, the at least one SC-β cell or precursor thereof aremaintained in culture by methods known by one of ordinary skill in theart, and in some embodiments, propagated prior to being converted intoSC-β cells by the methods as disclosed herein.

Further, at least one SC-β cell or precursor thereof, e.g., pancreaticprogenitor can be from any mammalian species, with non-limiting examplesincluding a murine, bovine, simian, porcine, equine, ovine, or humancell. For clarity and simplicity, the description of the methods hereinrefers to a mammalian at least one SC-β cell or precursor thereof but itshould be understood that all of the methods described herein can bereadily applied to other cell types of at least one SC-β cell orprecursor thereof. In some embodiments, the at least one SC-β cell orprecursor thereof is derived from a human individual.

Provided herein is a method for generating a stem cell-derived β (SC-β)cell comprising contacting a cell population comprising pancreaticprogenitor cells or precursors thereof with a histone deacetylase (HDAC)inhibitor to generate the SC-β cell, wherein the cell population isderived in vitro from stem cells. In some embodiments, the stem cellsare human pluripotent stem cells. In some embodiments, the methodfurther comprises contacting the cell population with at least one ofbetacellulin, thiazovinin, retinoic acid, SANT1, XXI, Alk5i II, GC-1,LDN, staurosporine, or any combination thereof. In some embodiments, theSC-β cell expresses C-PEP and NKX6-1. In some embodiments, the SC-β cellexhibits an in vitro glucose-stimulated insulin secretion response to aglucose challenge. In some embodiments, the method further comprisescontracting the cell population with a histone methyltransferaseinhibitor.

Provided herein is a method for generating a stem cell-derived β (SC-β)cell comprising contacting a cell population comprising pancreaticprogenitor cells or precursors thereof with a histone methyltransferaseinhibitor to generate the SC-β cell, wherein the cell population isderived in vitro from stem cells, and wherein the SC-β cell exhibits anin vitro glucose-stimulated insulin secretion response to a glucosechallenge. In some embodiments, the stem cells are human pluripotentstem cells. In some embodiments, the method further comprises contactingthe cell population with at least one of betacellulin, thiazovinin,retinoic acid, SANT1, XXI, Alk5i II, GC-1, LDN, staurosporine, or anycombination thereof. In some embodiments, the method further comprisescontacting the cell population with a histone deacetylase (HDAC)inhibitor.

Non-limiting exemplary epigenetic modifying compound include a DNAmethylation inhibitor, a histone acetyltransferase inhibitor, a histonedeacetylase inhibitor, a histone methyltransferase inhibitor, abromodomain inhibitor, or any combination thereof.

In an embodiment, the histone methyltransferase inhibitor is aninhibitor of enhancer of zeste homolog 2 (EZH2). EZH2 is ahistone-lysine N-methyltransferase enzyme. Non-limiting examples of anEZH2 inhibitor include 3-deazaneplanocin A (DZNep), EPZ6438, EPZ005687(an S-adenosylmethionine (SAM) competitive inhibitor), EI1, GSK126, andUNC1999. DZNep inhibits the hydrolysis of S-adenosyl-L-homocysteine(SAH), which is a product-based inhibitor of all proteinmethyltransferases, leading to increased cellular concentrations of SAHwhich in turn inhibits EZH2. DZNep is not specific to EZH2 and alsoinhibits other DNA methyltransferases. GSK126 is a SAM-competitive EZH2inhibitor that has 150-fold selectivity over EZH1. UNC1999 is ananalogue of GSK126, and it is less selective than its counterpartGSK126.

In an embodiment, the histone methyltransferase inhibitor is DZNep. Inan embodiment, the HDAC inhibitor is a class I HDAC inhibitor, a classII HDAC inhibitor, or a combination thereof. In an embodiment, thehistone methyltransferase inhibitor is KD5170 (mercaptoketone-based HDACinhibitor), MC1568 (class IIa HDAC inhibitor), TMP195 (class IIa HDACinhibitor), or any combination thereof. In some embodiments, HDACinhibitor is vorinostat, romidepsin (Istodax), chidamide, panobinostat(farydak), belinostat (PXD101), panobinostat (LBH589), valproic acid,mocetinostat (MGCD0103), abexinostat (PCI-24781), entinostat (MS-275),SB939, resminostat (4SC-201), givinostat (ITF2357), quisinostat(JNJ-26481585), HBI-8000, (a benzamide HDI), kevetrin, CUDC-101, AR-42,CHR-2845, CHR-3996, 4SC-202, CG200745, ACY-1215, ME-344, sulforaphane,or any variant thereof.

In some cases, the concentration of the histone methyltransferaseinhibitor (e.g., DZNep) can be from or from about 0.01 to 10 μM. Forexample, the concentration of the histone methyltransferase inhibitor(e.g., DZNep) can be about 0.01 to 1, 0.1 to 1, 0.25 to 1, 0.5 to 1, 1to 5; or 1 to 10 μM. The concentration of the histone methyltransferaseinhibitor (e.g., DZNep) can be less than about: 5, 4, 3, 2, 1.9, 1.8,1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3,0.2, 0.1, 0.05, or 0.01 μM.

Aspects of the disclosure involve definitive endoderm cells. Definitiveendoderm cells of use herein can be derived from any source or generatedin accordance with any suitable protocol. In some aspects, pluripotentstem cells, e.g., iPSCs or hESCs, are differentiated to endoderm cells.In some aspects, the endoderm cells (stage 1) are furtherdifferentiated, e.g., to primitive gut tube cells (stage 2),Pdx1-positive pancreatic progenitor cells (stage 3), NKX6.1-positivepancreatic progenitor cells (stage 4), or Ngn3-positive endocrineprogenitor cells or insulin-positive endocrine cells (stage 5), followedby induction or maturation to SC-β cells (stage 6).

In some cases, definitive endoderm cells can be obtained bydifferentiating at least some pluripotent cells in a population intodefinitive endoderm cells, e.g., by contacting a population ofpluripotent cells with i) at least one growth factor from the TGF-βsuperfamily, and ii) a WNT signaling pathway activator, to induce thedifferentiation of at least some of the pluripotent cells intodefinitive endoderm cells, wherein the definitive endoderm cells expressat least one marker characteristic of definitive endoderm.

Any growth factor from the TGF-β superfamily capable of inducing thepluripotent stem cells to differentiate into definitive endoderm cells(e.g., alone, or in combination with a WNT signaling pathway activator)can be used in the method provided herein. In some cases, the growthfactor from the TGF-β superfamily comprises Activin A. In some cases,the growth factor from the TGF-β superfamily comprises growthdifferentiating factor 8 (GDF8). Any WNT signaling pathway activatorcapable of inducing the pluripotent stem cells to differentiate intodefinitive endoderm cells (e.g., alone, or in combination with a growthfactor from the TGF-β superfamily) can be used in the method providedherein. In some cases, the WNT signaling pathway activator comprisesCHIR99Q21. In some cases, the WNT signaling pathway activator comprisesWnt3a recombinant protein.

In some cases, differentiating at least some pluripotent cells in apopulation into definitive endoderm cells is achieved by a process ofcontacting a population of pluripotent cells with i) Activin A, and ii)CHIR99021 for a suitable period of time, e.g., about 2 days, about 3days, about 4 days, or about 5 days to induce the differentiation of atleast some of the pluripotent cells in the population into definitiveendoderm cells, wherein the definitive endoderm cells express at leastone marker characteristic of definitive endoderm.

In some examples, the method comprises differentiating pluripotent cellsinto definitive endoderm cells by contacting a population of pluripotentcells with a suitable concentration of the growth factor from the TGF-βsuperfamily (e.g., Activin A), such as, about 10 ng/mL, about 20 ng/mL,about 50 ng/mL, about 75 ng/mL, about 80 ng/mL, about 90 ng/mL, about 95ng/mL, about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130ng/mL, about 140 ng/mL, about 150 ng/mL, about 175 ng/mL, about 180ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL. In somecases, the method comprises use of about 100 ng/mL Activin A fordifferentiation of pluripotent cells into definitive endoderm cells. Insome cases, the method comprises use of about 200 ng/mL Activin A fordifferentiation of pluripotent cells into definitive endoderm cells.

In some examples, the method comprises differentiating pluripotent cellsinto definitive endoderm cells by contacting a population of pluripotentcells with a suitable concentration of the WNT signaling pathwayactivator (e.g., CHIR99021), such as, about 0.01 μM, about 0.05 μM,about 0.1 μM, about 0.2 μM, about 0.5 μM, about 0.8 μM, about 1 μM,about 1.5 μM, about 2 μM, about 2.5 μM, about 3 μM, about 3.5 μM, about4 μM, about 5 μM, about 8 μM, about 10 μM, about 12 μM, about 15 μM,about 20 μM, about 30 μM, about 50 μM, about 100 μM, or about 200 μM. Insome cases, the method comprises use of about 2 μM CHIR99021 fordifferentiation of pluripotent cells into definitive endoderm cells. Insome cases, the method comprises use of about 5 μM CHIR99021 fordifferentiation of pluripotent cells into definitive endoderm cells.

In some cases, a definitive endoderm cell produced by the methods asdisclosed herein expresses at least one marker selected from the groupconsisting of: Nodal, Tmprss2, Tmem30b, St14, Spink3, Sh3gl2, Ripk4,Rab1S, Npnt, Clic6, Cldn5, Cacna1b, Bnip1, Anxa4, Emb, FoxA1, Sox17, andRbm35a, wherein the expression of at least one marker is upregulated toby a statistically significant amount in the definitive endoderm cellrelative to the pluripotent stem cell from which it was derived. In somecases, a definitive endoderm cell produced by the methods as disclosedherein does not express by a statistically significant amount at leastone marker selected the group consisting of: Gata4, SPARC, AFP and Dab2relative to the pluripotent stem cell from which it was derived. In somecases, a definitive endoderm cell produced by the methods as disclosedherein does not express by a statistically significant amount at leastone marker selected the group consisting of: Zic1, Pax6, Flk1 and CD31relative to the pluripotent stem cell from which it was derived. In somecases, a definitive endoderm cell produced by the methods as disclosedherein has a higher level of phosphorylation of Smad2 by a statisticallysignificant amount relative to the pluripotent stem cell from which itwas derived. In some cases, a definitive endoderm cell produced by themethods as disclosed herein has the capacity to form gut tube in vivo.In some cases, a definitive endoderm cell produced by the methods asdisclosed herein can differentiate into a cell with morphologycharacteristic of a gut cell, and wherein a cell with morphologycharacteristic of a gut cell expresses FoxA2 and/or Claudin6, In somecases, a definitive endoderm cell produced by the methods as disclosedherein can be further differentiated into a cell of endoderm origin.

In some cases, a population of pluripotent stem cells are cultured inthe presence of at least one β cell differentiation factor prior to anydifferentiation or during the first stage of differentiation. One canuse any pluripotent stem cell, such as a human pluripotent stem cell, ora human iPS cell or any of pluripotent stem cell as discussed herein orother suitable pluripotent stem cells. In some cases, a β celldifferentiation factor as described herein can be present in the culturemedium of a population of pluripotent stem cells or may be added inbolus or periodically during growth (e.g. replication or propagation) ofthe population of pluripotent stem cells. In certain examples, apopulation of pluripotent stem cells can be exposed to at least one βcell differentiation factor prior to any differentiation. In otherexamples, a population of pluripotent stem cells may be exposed to atleast one β cell differentiation factor during the first stage ofdifferentiation.

Aspects of the disclosure involve primitive gut tube cells. Primitivegut tube cells of use herein can be derived from any source or generatedin accordance with any suitable protocol. In some aspects, definitiveendoderm cells are differentiated to primitive gut tube cells. In someaspects, the primitive gut tube cells are further differentiated, e.g.,to Pdx1-positive pancreatic progenitor cells, NKX6.1-positive pancreaticprogenitor cells, Ngn3-positive endocrine progenitor cells,insulin-positive endocrine cells, followed by induction or maturation toSC-β cells.

In some cases, primitive gut tube cells can be obtained bydifferentiating at least some definitive endoderm cells in a populationinto primitive gut tube cells, e.g., by contacting definitive endodermcells with at least one growth factor from the fibroblast growth factor(FGF) family, to induce the differentiation of at least some of thedefinitive endoderm cells into primitive gut tube cells, wherein theprimitive gut tube cells express at least one marker characteristic ofprimitive gut tube cells.

Any growth factor from the FGF family capable of inducing definitiveendoderm cells to differentiate into primitive gut tube cells (e.g.,alone, or in combination with other factors) can be used in the methodprovided herein. In some cases, the at least one growth factor from theFGF family comprises keratinocyte growth factor (KGF). In some cases,the at least one growth factor from the FGF family comprises FGF2. Insome cases, the at least one growth factor from the FGF family comprisesFGF8B. In some cases, the at least one growth factor from the FGF familycomprises FGF 10. In some cases, the at least one growth factor from theFGF family comprises FGF21.

In some cases, primitive gut tube cells can be obtained bydifferentiating at least some definitive endoderm cells in a populationinto primitive gut tube cells, e.g., by contacting definitive endodermcells with KGF for a certain period of time, e.g., about 1 day, about 2days, about 3 days, or about 4 days, to induce the differentiation of atleast some of the definitive endoderm cells into primitive gut tubecells.

In some cases, the method comprises differentiating definitive endodermcells into primitive gut tube cells by contacting definitive endodermcells with a suitable concentration of the growth factor from the FGFfamily (e.g., KGF), such as, about 10 ng/mL, about 20 ng/mL, about 50ng/mL, about 75 ng/mL, about 80 ng/mL, about 90 ng/mL, about 95 ng/mL,about 100 ng/mL, about 110 ng/mL, about 120 ng/mL, about 130 ng/mL,about 140 ng/mL, about 150 ng/mL, about 175 ng/mL, about 180 ng/mL,about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL. In some cases, themethod comprises use of about 50 ng/mL KGF for differentiation ofdefinitive endoderm cells into primitive gut tube cells. In some cases,the method comprises use of about 100 ng/mL KGF for differentiation ofdefinitive endoderm cells into primitive gut tube cells.

Aspects of the disclosure involve Pdx1-positive pancreatic progenitorcells. Pdx1-positive pancreatic progenitor cells of use herein can bederived from any source or generated in accordance with any suitableprotocol. In some aspects, primitive gut tube cells are differentiatedto Pdx1-positive pancreatic progenitor cells. In some aspects, thePdx1-positive pancreatic progenitor cells are further differentiated,e.g., NKX6.1-positive pancreatic progenitor cells, Ngn3-positiveendocrine progenitor cells, insulin-positive endocrine cells, followedby induction or maturation to SC-β cells,

In some aspects, Pdx1-positive pancreatic progenitor cells can beobtained by differentiating at least some primitive gut tube cells in apopulation into Pdx1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with i) at least one BMP signalingpathway inhibitor, ii) a growth factor from TGF-β superfamily, iii) atleast one growth factor from the FGF family, iv) at least one SHHpathway inhibitor, v) at least one retinoic acid (RA) signaling pathwayactivator; vi) at least one protein kinase C activator, and vii) ROCKinhibitor to induce the differentiation of at least some of theprimitive gut tube cells into Pdx1-positive pancreatic progenitor cells,wherein the Pdx1-positive pancreatic progenitor cells express Pdx1.

In some aspects, Pdx1-positive pancreatic progenitor cells can beobtained by differentiating at least some primitive gut tube cells in apopulation into Pdx1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with i) at least one BMP signalingpathway inhibitor, ii) a growth factor from TGF-β superfamily, iii) atleast one growth factor from the FGF family, iv) at least one SHHpathway inhibitor, v) at least one retinoic acid (RA) signaling pathwayactivator; and vi) at least one protein kinase C activator, to inducethe differentiation of at least some of the primitive gut tube cellsinto Pdx1-positive pancreatic progenitor cells, wherein thePdx1-positive pancreatic progenitor cells express Pdx1.

In some cases, Pdx1-positive pancreatic progenitor cells can be obtainedby differentiating at least some primitive gut tube cells in apopulation into Pdx1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with i) at least one BMP signalingpathway inhibitor, ii) at least one growth factor from the FGF family,iii) at least one SHH pathway inhibitor, iv) at least one retinoic acid(RA) signaling pathway activator; and v) at least one protein kinase Cactivator, to induce the differentiation of at least some of theprimitive gut tube cells into Pdx1-positive pancreatic progenitor cells,wherein the Pdx1-positive pancreatic progenitor cells express Pdx1.

In some cases, Pdx1-positive pancreatic progenitor cells can be obtainedby differentiating at least some primitive gut tube cells in apopulation into Pdx1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with i) at least one SHH pathwayinhibitor, ii) at least one retinoic acid (RA) signaling pathwayactivator; and iii) at least one protein kinase C activator, wherein thePdx1-positive pancreatic progenitor cells express Pdx1.

In some cases, Pdx1-positive pancreatic progenitor cells can be obtainedby differentiating at least some primitive gut tube cells in apopulation into Pdx1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with i) at least one growth factorfrom the FGF family, and ii) at least one retinoic acid (RA) signalingpathway activator, to induce the differentiation of at least some of theprimitive gut tube cells into Pdx1-positive pancreatic progenitor cells,wherein the Pdx1-positive pancreatic progenitor cells express Pdx1.

Any BMP signaling pathway inhibitor capable of inducing primitive guttube cells to differentiate into Pdx1-positive pancreatic progenitorcells (e.g., alone, or with any combination of a growth factor fromTGF-β superfamily, at least one growth factor from the FGF family, atleast one SHH pathway inhibitor, at least one retinoic acid signalingpathway activator, at least one protein kinase C activator, and ROCKinhibitor) can be used in the method provided herein. In some cases, theBMP signaling pathway inhibitor comprises LDN193189 or DMH-1. In someexamples, the method comprises contacting primitive gut tube cells witha concentration of BMP signaling pathway inhibitor (e.g., LDN1931189),such as, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM,about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM,about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM,about 230 nM, about 240 nM, about 250 nM, about 280 nM, about 300 nM,about 400 nM, about 500 nM, or about 1 μM. In some examples, the methodcomprises contacting primitive gut tube cells with a concentration ofBMP signaling pathway inhibitor (e.g., DMH-1), such as, about 0.01 μM,about 0.02 μM, about 0.05 μM, about 0.1 μM, about 0.2 μM, about 0.5 μM,about 0.8 μM, about 1 μM, about 1.2 μM, about 1.5 μM, about 1.75 μM,about 2 μM, about 2.2 μM, about 2.5 μM, about 2.75 μM, about 3 μM, about3.25 μM, about 3.5 μM, about 3.75 μM, about 4 μM, about 4.5 μM, about 5μM, about 8 μM, about 10 μM, about 15 μM, about 20 μM, about 30 μM,about 40 μM, about 50 μM, or about 100 μM.

Any growth factor from the TGF-β superfamily capable of inducingprimitive gut tube cells to differentiate into Pdx1-positive pancreaticprogenitor cells (e.g., alone, or with any combination of at least oneBMP signaling pathway inhibitor, a growth factor from the FGF family, atleast one SHH pathway inhibitor, at least one retinoic acid signalingpathway activator, at least one protein kinase C activator, and ROCKinhibitor) can be used. In some cases, the growth factor from TGF-βfamily comprises Activin A. In some cases, the growth factor from TGF-βfamily comprises Activin A or GDF8. In some examples, the methodcomprises contacting primitive gut tube cells with a concentration of agrowth factor from TGF-β superfamily (e.g., Activin A), such as, about 5ng/mL, about 7.5 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL,about 11 ng/mL, about 12 ng/mL, about 13 ng/mL, about 14 ng/mL, about 15ng/mL, about 16 ng/mL, about 17 ng/mL, about 18 ng/mL, about 19 ng/mL,about 20 ng/mL, about 21 ng/mL, about 22 ng/mL, about 23 ng/mL, about 24ng/mL, about 25 ng/mL, about 26 ng/mL, about 27 ng/mL, about 28 ng/mL,about 29 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50ng/mL, or about 100 ng/mL.

Any growth factor from the FGF family capable of inducing primitive guttube cells to differentiate into Pdx1-positive pancreatic progenitorcells (e.g., alone, or with any combination of at least one BMPsignaling pathway inhibitor, a growth factor from TGF-β superfamily, atleast one SHH pathway inhibitor, at least one retinoic acid signalingpathway activator, at least one protein kinase C activator, and ROCKinhibitor) can be used. In some cases, the at least one growth factorfrom the FGF family comprises keratinocyte growth factor (KGF). In somecases, the at least one growth factor from the FGF family is selectedfrom the group consisting of FGF2, FGF8B, FGF 10, and FGF21. In someexamples, the method comprises contacting primitive gut tube cells witha concentration of a growth factor from FGF family (e.g., KGF), such as,about 10 ng/mL, about 20 ng/mL, about 50 ng/mL, about 75 ng/mL, about 80ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 110 ng/mL,about 120 ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL,about 175 ng/mL, about 180 ng/mL, about 200 ng/mL, about 250 ng/mL, orabout 300 ng/mL.

Any SHH pathway inhibitor capable of inducing primitive gut tube cellsto differentiate into Pdx1-positive pancreatic progenitor cells (e.g.,alone, or with any combination of at least one BMP signaling pathwayinhibitor, at least one growth factor from the FGF family, a growthfactor from TGF-β superfamily, at least one retinoic acid signalingpathway activator, at least one protein kinase C activator, and ROCKinhibitor) can be used. In some cases, the SHH pathway inhibitorcomprises Sant1. In some examples, the method comprises contactingprimitive gut tube cells with a concentration of a SHH pathway inhibitor(e.g., Sant1), such as, about 0.001 μM, about 0.002 μM, about 0.005 μM,about 0.01 μM, about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08μM, about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about0.15 μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM,about 0.2 μM, about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24μM, about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about0.29 μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM,about 0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM,about 0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM.

Any RA signaling pathway activator capable of inducing primitive guttube cells to differentiate into Pdx1-positive pancreatic progenitorcells (e.g., alone, or with any combination of at least one BMPsignaling pathway inhibitor, at least one growth factor from the FGFfamily, at least one SHH pathway inhibitor, at least one protein kinaseC activator, and ROCK inhibitor) can be used. In some cases, the RAsignaling pathway activator comprises retinoic acid. In some examples,the method comprises contacting primitive gut tube cells with aconcentration of an RA signaling pathway activator (e.g., retinoicacid), such as, about 0.02 μM, about 0.1 μM, about 0.2 μM, about 0.25μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM, about 0.8μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM,about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM,about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM,about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM,about 2.8 μM, about 3 μM, about 3.2 μM, about 3.4 μM, about 3.6 μM,about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM,about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM,about 10 μM, about 12 μM, about 14 μM, about 15 μM, about 16 μM, about18 μM, about 20 μM, about 50 μM, or about 100 μM.

Any PKC activator capable of inducing primitive gut tube cells todifferentiate into Pdx1-positive pancreatic progenitor cells (e.g.,alone, or with any combination of at least one BMP signaling pathwayinhibitor, at least one growth factor from the FGF family, at least oneSHH pathway inhibitor, at least one RA signaling pathway activator, andROCK inhibitor) can be used. In some cases, the PKC activator comprisesPdBU. In some cases, the PKC activator comprises TPB. In some examples,the method comprises contacting primitive gut tube cells with aconcentration of a PKC activator (e.g., PdBU), such as, about 10 μM,about 20 μM, about 50 μM, about 75 μM, about 80 μM, about 100 μM, about120 μM, about 140 μM, about 150 μM, about 175 μM, about 180 μM, about200 μM, about 210 μM, about 220 μM, about 240 μM, about 250 μM, about260 μM, about 280 μM, about 300 μM, about 320 μM, about 340 μM, about360 μM, about 380 μM, about 400 μM, about 420 μM, about 440 μM, about460 μM, about 480 μM, about 500 μM, about 520 μM, about 540 μM, about560 μM, about 580 μM, about 600 μM, about 620 μM, about 640 μM, about660 μM, about 680 μM, about 700 μM, about 750 μM, about 800 μM, about850 μM, about 900 μM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, orabout 5 mM.

Any ROCK inhibitor capable of inducing primitive gut tube cells todifferentiate into Pdx1-positive pancreatic progenitor cells (e.g.,alone, or with any combination of at least one BMP signaling pathwayinhibitor, at least one growth factor from the FGF family, at least oneSHH pathway inhibitor, PKC activator, and at least one RA signalingpathway activator) can be used. In some cases, the ROCK inhibitorcomprises Thiazovivin, Y-27632, Fasudil/HA1077, or H-1152. In somecases, the ROCK inhibitor comprises Y-27632. In some cases, the ROCKinhibitor comprises Thiazovivin. In some examples, the method comprisescontacting primitive gut tube cells with a concentration of a ROCKinhibitor (e.g., Y-27632 or Thiazovivin), such as, about 0.2 μM, about0.5 μM, about 0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM,about 5 μM, about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM,about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, about20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM,about 26 μM, about 27 μM, about 28 μM, about 29 μM, about 30 μM, about35 μM, about 40 μM, about 50 μM, or about 100 μM.

In some cases, Pdx1-positive pancreatic progenitor cells can be obtainedby differentiating at least some primitive gut tube cells in apopulation into Pdx1-positive pancreatic progenitor cells, e.g., bycontacting primitive gut tube cells with retinoic acid, KGF, Sant1,LDN193189, PdBU, Y-27632, and Activin A, for a suitable period of time,e.g., about 1 day, about 2 days, about 3 days, or about 4 days. In somecases, Pdx1-positive pancreatic progenitor cells can be obtained bydifferentiating at least some primitive gut tube cells in a populationinto Pdx1-positive pancreatic progenitor cells, e.g., by contactingprimitive gut tube cells with retinoic acid, KGF, Sant1, LDN193189,PdBU, Y-27632, and Activin A, for about 2 days. In some cases,Pdx1-positive pancreatic progenitor cells can be obtained bydifferentiating at least some primitive gut tube cells in S3 medium.

Aspects of the disclosure involve NKX6.1-positive pancreatic progenitorcells. NKX6.1-positive pancreatic progenitor cells of use herein can bederived from any source or generated in accordance with any suitableprotocol. In some aspects, Pdx1-positive pancreatic progenitor cells aredifferentiated to NKX6.1-positive pancreatic progenitor cells. In someaspects, the NKX6.1-positive pancreatic progenitor cells are furtherdifferentiated, e.g., to Ngn3-positive endocrine progenitor cells, orinsulin-positive endocrine cells, followed by induction or maturation toSC-β cells.

In some aspects, a method of producing a NKX6.1-positive pancreaticprogenitor cell from a Pdx1-positive pancreatic progenitor cellcomprises contacting a population of cells (e.g., under conditions thatpromote cell clustering and/or promoting cell survival) comprisingPdx1-positive pancreatic progenitor cells with at least two βcell-differentiation factors comprising a) at least one growth factorfrom the fibroblast growth factor (FGF) family, b) a sonic hedgehogpathway inhibitor, and optionally c) a low concentration of a retinoicacid (RA) signaling pathway activator, to induce the differentiation ofat least one Pdx1-positive pancreatic progenitor cell in the populationinto NKX6.1-positive pancreatic progenitor cells, wherein theNKX6.1-positive pancreatic progenitor cells expresses NKX6.1.

In some cases, the Pdx1-positive, NKX6.1-positive pancreatic progenitorcells are obtained by contacting Pdx1-positive pancreatic progenitorcells with i) at least one growth factor from the FGF family, ii) atleast one SHH pathway inhibitor, and optionally iii) a low concentrationof a RA signaling pathway activator, to induce the differentiation of atleast some of the Pdx1-positive pancreatic progenitor cells intoPdx1-positive, NKX6.1-positive pancreatic progenitor cells, wherein thePdx1-positive, NKX6.1-positive pancreatic progenitor cells expressesPdx1 and NKX6.1.

In some cases, the Pdx1-positive, NKX6.1-positive pancreatic progenitorcells are obtained by contacting Pdx1-positive pancreatic progenitorcells with i) at least one growth factor from the FGF family, ii) atleast one SHH pathway inhibitor, and optionally iii) a low concentrationof a RA signaling pathway activator, iv) ROCK inhibitor, and v) at leastone growth factor from the TGF-β superfamily, to induce thedifferentiation of at least some of the Pdx1-positive pancreaticprogenitor cells into Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells. In some cases, the Pdx1-positive, NKX6.1-positivepancreatic progenitor cells are obtained by contacting Pdx1-positivepancreatic progenitor cells under conditions that promote cellclustering with at least one growth factor from the FGF family.

In some cases, the Pdx1-positive pancreatic progenitor cells areproduced from a population of pluripotent cells. In some cases, thePdx1-positive pancreatic progenitor cells are produced from a populationof iPS cells. In some cases, the Pdx1-positive pancreatic progenitorcells are produced from a population of ESC cells. In some cases, thePdx1-positive pancreatic progenitor cells are produced from a populationof definitive endoderm cells. In some cases, the Pdx1-positivepancreatic progenitor cells are produced from a population of primitivegut tube cells.

Any growth factor from the FGF family capable of inducing Pdx1-positivepancreatic-progenitor cells to differentiate into NKX6.1-positivepancreatic progenitor cells (e.g., alone, or with any combination of atleast one SHH pathway inhibitor, a ROCK inhibitor, a growth factor fromthe TGF-β superfamily, and at least one retinoic acid signaling pathwayactivator) can be used in the method provided herein. In some cases, theat least one growth factor from the FGF family comprises keratinocytegrowth factor (KGF). In some cases, the at least one growth factor fromthe FGF family is selected from the group consisting of FGF2, FGF8B, FGF10, and FGF21. In some examples, the method comprises contactingPdx1-positive pancreatic progenitor cells with a concentration of agrowth factor from FGF family (e.g., KGF), such as, about 10 ng/mL,about 20 ng/mL, about 50 ng/mL, about 75 ng/mL, about 80 ng/mL, about 90ng/mL, about 95 ng/mL, about 100 ng/mL, about 110 ng/mL, about 120ng/mL, about 130 ng/mL, about 140 ng/mL, about 150 ng/mL, about 175ng/mL, about 180 ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300ng/mL.

Any SHH pathway inhibitor capable of inducing Pdx1-positive pancreaticprogenitor cells to differentiate into NKX6.1-positive pancreaticprogenitor cells (e.g., alone, or with any combination of at least onegrowth factor from the FGF family, at least one retinoic acid signalingpathway activator, ROCK inhibitor, and at least one growth factor fromthe TGF-β superfamily) can be used in the method provided herein. Insome cases, the SHH pathway inhibitor comprises Sant1. In some examples,the method comprises contacting Pdx1-positive pancreatic progenitorcells with a concentration of a SHH pathway inhibitor (e.g., Sant1),such as, about 0.001 μM, about 0.002 μM, about 0.005 μM, about 0.01 μM,about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08 μM, about 0.1μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15 μM, about0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about 0.2 μM,about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24 μM, about 0.25μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29 μM, about0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about 0.34 μM,about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.6 μM,about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM.

Any RA signaling pathway activator capable of inducing Pdx1-positivepancreatic progenitor cells to differentiate into NKX6.1-positivepancreatic progenitor cells (e.g., alone, or with any combination of atleast one growth factor from the FGF family, at least one SHH pathwayinhibitor, ROCK inhibitor, and at least one growth factor from the TGF-βsuperfamily) can be used. In some cases, the RA signaling pathwayactivator comprises retinoic acid. In some examples, the methodcomprises contacting Pdx1-positive pancreatic progenitor cells with aconcentration of an RA signaling pathway activator (e.g., retinoicacid), such as, about 0.02 μM, about 0.1 μM, about 0.2 μM, about 0.25μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about 0.55μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM, about 0.8μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM, about 1.2 μM,about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM,about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM, about 2.2 μM,about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM, about 2.7 μM,about 2.8 μM, about 3 μM, about 3.2 μM, about 3.4 μM, about 3.6 μM,about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM, about 4.6 μM,about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about 6.5 μM, about7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM, about 9.5 μM,about 10 μM, about 12 μM, about 14 μM, about 15 μM, about 16 μM, about18 μM, about 20 μM, about 50 μM, or about 100 μM.

Any ROCK inhibitor capable of inducing Pdx1-positive pancreaticprogenitor cells to differentiate into NKX6.1-positive pancreaticprogenitor cells (e.g., alone, or with any combination of at least onegrowth factor from the FGF family, at least one SHH pathway inhibitor, aRA signaling pathway activator, and at least one growth factor from theTGF-β superfamily) can be used. In some cases, the ROCK inhibitorcomprises Thiazovivin, Y-27632, Fasudil/HA1077, or 14-1152. In someexamples, the method comprises contacting Pdx1-positive pancreaticprogenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632or Thiazovivin), such as, about 0.2 μM, about 0.5 μM, about 0.75 μM,about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM,about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 11μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM,about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM,about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about50 μM, or about 100 μM.

Any activator from the TGF-β superfamily capable of inducingPdx1-positive pancreatic progenitor cells to differentiate intoNKX6.1-positive pancreatic progenitor cells (e.g., alone, or with anycombination of at least one growth factor from the FGF family, at leastone SHH pathway inhibitor, a RA signaling pathway activator, and ROCKinhibitor) can be used. In some cases, the activator from the TGF-βsuperfamily comprises Activin A or GDF8. In some examples, the methodcomprises contacting Pdx1-positive pancreatic progenitor cells with aconcentration of a growth factor from TGF-β superfamily (e.g., ActivinA), such as, about 0.1 ng/mL, about 0.2 ng/mL, about 0.3 ng/mL, about0.4 ng/mL, about 0.5 ng/mL, about 0.6 ng/mL, about 0.7 ng/mL, about 0.8ng/mL, about 1 ng/mL, about 1.2 ng/mL, about 1.4 ng/mL, about 1.6 ng/mL,about 1.8 ng/mL, about 2 ng/mL, about 2.2 ng/mL, about 2.4 ng/mL, about2.6 ng/mL, about 2.8 ng/mL, about 3 ng/mL, about 3.2 ng/mL, about 3.4ng/mL, about 3.6 ng/mL, about 3.8 ng/mL, about 4 ng/mL, about 4.2 ng/mL,about 4.4 ng/mL, about 4.6 ng/mL, about 4.8 ng/mL, about 5 ng/mL, about5.2 ng/mL, about 5.4 ng/mL, about 5.6 ng/mL, about 5.8 ng/mL, about 6ng/mL, about 6.2 ng/mL, about 6.4 ng/mL, about 6.6 ng/mL, about 6.8ng/mL, about 7 ng/mL, about 8 ng/mL, about 9 ng/mL, about 10 ng/mL,about 20 ng/mL, about 30 ng/mL, or about 50 ng/mL. In some examples, themethod comprises contacting Pdx1-positive pancreatic progenitor cellswith a concentration of a growth factor from TGF-β superfamily (e.g.,Activin A), such as, about 5 ng/mL.

In some cases, the Pdx1-positive, NKX6.1-positive pancreatic progenitorcells are obtained by contacting Pdx1-positive pancreatic progenitorcells under conditions that promote cell clustering with KGF, Sant1, andRA, for a period of 5 days. In some cases, the Pdx1-positive,NKX6.1-positive pancreatic progenitor cells are obtained by contactingPdx1-positive pancreatic progenitor cells under conditions that promotecell clustering with KGF, Sant1, RA, Y27632, and Activin A, for a periodof 5 days. In some cases, the Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells are obtained by contacting Pdx1-positive pancreaticprogenitor cells under conditions that promote cell clustering with KGFfor a period of 5 days. In some cases, the Pdx1-positive,NKX6.1-positive pancreatic progenitor cells are obtained by contactingPdx1-positive pancreatic progenitor cells in a S3 medium.

Aspects of the disclosure involve insulin-positive endocrine cells.Insulin-positive endocrine cells of use herein can be derived from anysource or generated in accordance with any suitable protocol, In someaspects, NKX6.1-positive pancreatic progenitor cells are differentiatedto insulin-positive endocrine cells, In some aspects, theinsulin-positive endocrine cells are further differentiated, e.g., byinduction or maturation to SC-β cells.

In some aspects, a method of producing an insulin-positive endocrinecell from an NKX6.1-positive pancreatic progenitor cell comprisescontacting a population of cells (e.g., under conditions that promotecell clustering) comprising NKX6-1-positive pancreatic progenitor cellswith a) a TGF-β signaling pathway inhibitor, and b) a thyroid hormonesignaling pathway activator, to induce the differentiation of at leastone NKX6.1-positive pancreatic progenitor cell in the population into aninsulin-positive endocrine cell, wherein the insulin-positive endocrineceil expresses insulin. In some cases, insulin-positive endocrine cellsexpress Pdx1, NKX6.1, NKX2.2, Mafb, glis3, Sur1, Kir6.2, Znt8, SLC2A1,SLC2A3 and/or insulin.

Any TGF-β signaling pathway inhibitor capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells todifferentiate into insulin-positive endocrine cells (e.g., alone, or incombination with other β cell-differentiation factors, e.g., a thyroidhormone signaling pathway activator) can be used. In some cases, theTGF-β signaling pathway comprises TGF-β receptor type I kinasesignaling. In some cases, the TGF-β signaling pathway inhibitorcomprises Alk5 inhibitor II.

Any thyroid hormone signaling pathway activator capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells todifferentiate into insulin-positive endocrine cells (e.g., alone, or incombination with other 13 cell-differentiation factors, e.g., a TGF-βsignaling pathway inhibitor) can be used. In some cases, the thyroidhormone signaling pathway activator comprises triiodothyronine (T3). Insome cases, the thyroid hormone signaling pathway activator comprisesGC-1.

In some cases, the method comprises contacting the population of cells(e.g., NKX6.1-positive pancreatic progenitor cells) with at least oneadditional factor. In some cases, the method comprises contacting thePdx1-positive NKX6.1-positive pancreatic progenitor cells with at leastone of i) a SHH pathway inhibitor, ii) a RA signaling pathway activator,iii) a γ-secretase inhibitor, iv) at least one growth factor from theepidermal growth factor (EGF) family, v) a protein kinase inhibitor, vi)a TGF-β signaling pathway inhibitor, or vii) a thyroid hormone signalingpathway activator.

In some cases, the method comprises contacting the Pdx1-positiveNKX6.1-positive pancreatic progenitor cells with at least one of i) aSHH pathway inhibitor, ii) a RA signaling pathway activator, iii) aγ-secretase inhibitor, iv) at least one growth factor from the epidermalgrowth factor (EGF) family, v) at least one bone morphogenetic protein(BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathwayinhibitor, vii) a thyroid hormone signaling pathway activator, viii) aprotein kinase inhibitor, or ix) a ROCK inhibitor.

In some cases, the method comprises contacting the Pdx1-positiveNKX6.1-positive pancreatic progenitor cells with at least one of i) aSHH pathway inhibitor, ii) a RA signaling pathway activator, iii) aγ-secretase inhibitor, iv) at least one growth factor from the epidermalgrowth factor (EGF) family, v) at least one bone morphogenetic protein(BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathwayinhibitor, vii) a thyroid hormone signaling pathway activator, viii) anepigenetic modifying compound, ix) a protein kinase inhibitor, or x) aROCK inhibitor.

In some embodiments, in the method of generating the insulin-positiveendocrine cells from the Pdx1-positive NKX6.1-positive pancreaticprogenitor cells, some of the differentiation factors are present onlyfor the first 1, 2, 3, 4, or 5 days during the differentiation step. Insome cases, some of the differentiation factors, such as the SHH pathwayinhibitor, the RA signaling pathway activator, and the at least onegrowth factor from the EGF family are removed from the culture mediumafter the first 3 days of incubation.

Any γ-secretase inhibitor that is capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells in apopulation into insulin-positive endocrine cells (e.g., alone, or incombination with any of a TGF-β signaling pathway inhibitor and/or athyroid hormone signaling pathway activator). In some cases, theγ-secretase inhibitor comprises XXI. In some cases, the γ-secretaseinhibitor comprises DAPT. In some examples, the method comprisescontacting NKX6.1-positive pancreatic progenitor cells with aconcentration of a γ-secretase inhibitor (e.g., XXI), such as, about0.01 μM, about 0.02 μM, about 0.05 μM, about 0.075 μM, about 0.1 μM,about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM,about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1 μM, about 1.1 μM,about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM,about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM,about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM,about 2.7 μM, about 2.8 μM, about 2.9 μM, about 3 μM, about 3.2 μM,about 3.4 μM, about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM,about 4.4 μM, about 4.6 μM, about 4.8 μM, about 5 μM, about 5.2 μM,about 5.4 μM, about 5.6 μM, about 5.8 μM, about 6 μM, about 6.2 μM,about 6.4 μM, about 6.6 μM, about 6.8 μM, about 7 μM, about 8 μM, about9 μM, about 10 μM, about 20 μM, about 30 μM, or about 50 μM.

Any growth factor from the EGF family capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells in apopulation into insulin-positive endocrine cells (e.g., alone, or incombination with any of a TGF-β signaling pathway inhibitor and/or athyroid hormone signaling pathway activator) can be used. In some cases,the at least one growth factor from the EG F family comprisesbetacellulin. In some cases, at least one growth factor from the EGFfamily comprises EGF. In some examples, the method comprises contactingNKX6.1-positive pancreatic progenitor cells with a concentration of agrowth factor from EGF family (e.g., betacellulin), such as, about 1ng/mL, about 2 ng/mL, about 4 ng/mL, about 6 ng/mL, about 8 ng/mL, about10 ng/mL, about 12 ng/mL, about 14 ng/mL, about 16 ng/mL, about 18ng/mL, about 20 ng/mL, about 22 ng/mL, about 24 ng/mL, about 26 ng/mL,about 28 ng/mL, about 30 ng/mL, about 40 ng/mL, about 50 ng/mL, about 75ng/mL, about 80 ng/mL, about 90 ng/mL, about 95 ng/mL, about 100 ng/mL,about 150 ng/mL, about 200 ng/mL, about 250 ng/mL, or about 300 ng/mL.

Any RA signaling pathway activator capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells todifferentiate into insulin-positive endocrine cells (e.g., alone, or incombination with any of a TGF-β signaling pathway inhibitor and/or athyroid hormone signaling pathway activator) can be used. In some cases,the RA signaling pathway activator comprises RA. In some examples, themethod comprises contacting NKX6.1-positive pancreatic progenitor cellswith a concentration of an RA signaling pathway activator (e.g.,retinoic acid), such as, about 0.02 μM, about 0.1 μM, about 0.2 μM,about 0.25 μM, about 0.3 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM,about 0.55 μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM,about 0.8 μM, about 0.85 μM, about 0.9 μM, about 1 μM, about 1.1 μM,about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM,about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2 μM, about 2.1 μM,about 2.2 μM, about 2.3 μM, about 2.4 μM, about 2.5 μM, about 2.6 μM,about 2.7 μM, about 2.8 μM, about 3 μM, about 3.2 μM, about 3.4 μM,about 3.6 μM, about 3.8 μM, about 4 μM, about 4.2 μM, about 4.4 μM,about 4.6 μM, about 4.8 μM, about 5 μM, about 5.5 μM, about 6 μM, about6.5 μM, about 7 μM, about 7.5 μM, about 8 μM, about 8.5 μM, about 9 μM,about 9.5 μM, about 10 μM, about 12 μM, about 14 μM, about 15 μM, about16 μM, about 18 μM, about 20 μM, about 50 μM, or about 100 μM.

Any SHH pathway inhibitor capable of inducing the differentiation ofNKX6.1-positive pancreatic progenitor cells to differentiate intoinsulin-positive endocrine cells (e.g., alone, or in combination withany of a TGF-β signaling pathway inhibitor and/or a thyroid hormonesignaling pathway activator) can be used in the method provided herein.In some cases, the SHH pathway inhibitor comprises Sant1. In someexamples, the method comprises contacting NKX6.1-positive pancreaticprogenitor cells with a concentration of a SHH pathway inhibitor (e.g.,Sant1), such as, about 0.001 μM, about 0.002 μM, about 0.005 μM, about0.01 μM, about 0.02 μM, about 0.03 μM, about 0.05 μM, about 0.08 μM,about 0.1 μM, about 0.12 μM, about 0.13 μM, about 0.14 μM, about 0.15μM, about 0.16 μM, about 0.17 μM, about 0.18 μM, about 0.19 μM, about0.2 μM, about 0.21 μM, about 0.22 μM, about 0.23 μM, about 0.24 μM,about 0.25 μM, about 0.26 μM, about 0.27 μM, about 0.28 μM, about 0.29μM, about 0.3 μM, about 0.31 μM, about 0.32 μM, about 0.33 μM, about0.34 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM, about0.6 μM, about 0.8 μM, about 1 μM, about 2 μM, or about 5 μM.

Any BMP signaling pathway inhibitor capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells todifferentiate into insulin-positive endocrine cells (e.g., alone, or incombination with any of a TGF-β signaling pathway inhibitor and/or athyroid hormone signaling pathway activator) can be used. In some cases,the BMP signaling pathway inhibitor comprises LDN193189 or DMH-1. Insome examples, the method comprises contacting NKX6.1-positivepancreatic progenitor cells with a concentration of BMP signalingpathway inhibitor (e.g., LDN1931189), such as, about 30 nM, about 40 nM,about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about250 nM, about 280 nM, about 300 nM, about 400 nM, about 500 nM, or about1 μM.

Any ROCK inhibitor that is capable of inducing the differentiation ofNKX6.1-positive pancreatic progenitor cells in a population intoinsulin-positive endocrine cells (e.g., alone, or in combination withany of a TGF-β signaling pathway inhibitor and/or a thyroid hormonesignaling pathway activator) can be used. In some cases, the ROCKinhibitor comprises Thiazovivin, Y-27632, Fasudil/HA1077, or H-1152. Insome cases, the ROCK inhibitor comprises Y-27632. In some cases, theROCK inhibitor comprises Thiazovivin. In some examples, the methodcomprises contacting Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells with a concentration of a ROCK inhibitor (e.g., Y-27632or Thiazovivin), such as, about 0.2 μM, about 0.5 μM, about 0.75 μM,about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM,about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10 μM, about 11μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM,about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about22 μM, about 23 μM, about 24 μM, about 25 μM, about 26 μM, about 27 μM,about 28 μM, about 29 μM, about 30 μM, about 35 μM, about 40 μM, about50 μM, or about 100 μM.

Any epigenetic modifying compound that is capable of inducing thedifferentiation of NKX6.1-positive pancreatic progenitor cells in apopulation into insulin-positive endocrine cells (e.g., alone, or incombination with any of a TGF-β signaling pathway inhibitor and/or athyroid hormone signaling pathway activator) can be used. In some cases,the epigenetic modifying compound comprises a histone methyltransferaseinhibitor or a HDAC inhibitor. In some cases, the epigenetic modifyingcompound comprises a histone methyltransferase inhibitor, e.g., DZNep.In some cases, the epigenetic modifying compound comprises a HDACinhibitor, e.g., KD5170. In some examples, the method comprisescontacting Pdx1-positive, NKX6.1-positive pancreatic progenitor cellswith a concentration of an epigenetic modifying compound (e.g., DZNep orKD5170), such as, about 0.01 μM, about 0.025 μM, about 0.05 μM, about0.075 μM, about 0.1 μM, about 0.15 μM, about 0.2 μM, about 0.5 μM, about0.75 μM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM,about 6 μM, about 7 μM, about 7.5 μM, about 8 μM, about 9 μM, about 10μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM,about 40 μM, about 50 μM, or about 100 μM.

In some cases, the population of cells is optionally contacted with aprotein kinase inhibitor. In some cases, the population of cells is notcontacted with the protein kinase inhibitor. In some cases, thepopulation of cells is contacted with the protein kinase inhibitor. Anyprotein kinase inhibitor that is capable of inducing the differentiationof NKX6.1-positive pancreatic progenitor cells in a population intoinsulin-positive endocrine cells (e.g., alone, or in combination withany of a TGF-β signaling pathway inhibitor and/or a thyroid hormonesignaling pathway activator). In some cases, the protein kinaseinhibitor comprises staurosporine.

In some cases, the method comprises contacting the population of cells(e.g., NKX6.1-positive pancreatic progenitor cells) with XXI, Alk5i, T3or GC-1, RA, Sant1, and betacellulin for a period of 7 days, to inducethe differentiation of at least one NKX6.1-positive pancreaticprogenitor cell in the population into an insulin-positive endocrinecell, wherein the insulin-positive endocrine cell expresses insulin. Insome cases, the method comprises contacting the population of cells(e.g., NKX6.1-positive pancreatic progenitor cells) with XXI, Alk5i, T3or GC-1, RA, Sant1, betacellulin, and LDN193189 for a period of 7 days,to induce the differentiation of at least one NKX6.1-positive pancreaticprogenitor cell in the population into an insulin-positive endocrinecell, wherein the insulin-positive endocrine ceil expresses insulin. Insome embodiments, one or more differentiation factors are added in aportion of the Stage 5, for instance, only the first 1, 2, 3, 4, 5, or 6days of the period of time for Stage 5, or the last 1, 2, 3, 4, 5, or 6days of the period of time for Stage 5. In one example, the cells arecontacted with SHH signaling pathway inhibitor for only the first 2, 3,4, or 5 days during Stage 5, after which the SHH signaling pathwayinhibitor is removed from the culture medium. In another example, thecells are contacted with BMP signaling pathway inhibitor for only thefirst 1, 2, or 3 days during Stage 5, after which the BMP signalingpathway inhibitor is removed from the culture medium.

In some cases, the method comprises culturing the population of cells(e.g., NKX6.1-positive pancreatic progenitor cells) in a BE5 medium, toinduce the differentiation of at least one NKX6.1-positive pancreaticprogenitor cell in the population into an insulin-positive endocrinecell, wherein the insulin-positive endocrine cell expresses insulin.

Aspects of the disclosure involve generating pancreatic β cells (e.g.,non-native pancreatic β cells). Non-native pancreatic β cells, in somecases, resemble endogenous mature β cells in form and function, butnevertheless are distinct from native β cells.

In some cases, the insulin-positive pancreatic endocrine cells generatedusing the method provided herein can form a cell cluster, alone ortogether with other types of cells, e.g., precursors thereof, e.g., stemcell, definitive endoderm cells, primitive gut tube cell, Pdx1-positivepancreatic progenitor cells, or NKX6.1-positive pancreatic progenitorcells.

In some cases, the cell population comprising the insulin-positiveendocrine cells can be directly induced to mature into SC-β cellswithout addition of any exogenous differentiation factors (such asinhibitor of TGF-β signaling pathway, thyroid hormone signaling pathwayactivator, PKC activator, growth factors from TGF-β superfamily, FGFfamily, or EGF family, SHH signaling pathway inhibitor, γ-secretaseinhibitor, ROCK inhibitor, or BMP signaling pathway inhibitor).

In some cases, the cell population comprising the insulin-positiveendocrine cells can be directly induced to mature into SC-β cells bycontacting the insulin-positive endocrine cells with differentiationfactors. The differentiation factors can comprise at least one inhibitorof TGF-β signaling pathway and thyroid hormone signaling pathwayactivator as described herein. In some cases, SC-β cells can be obtainedby contacting a population of cells comprising insulin-positiveendocrine cells with Alk5i and T3 or GC-1.

In some examples, insulin-positive endocrine cells can be matured in aNS-GFs medium, MCDB131 medium, DMEM medium, or CMRL medium. In somecases, the insulin-positive endocrine cells can be matured in a CMRLsmedium supplemented with 10% FBS. In some cases, the insulin-positiveendocrine cells can be matured in a DMEM medium supplemented with 1%HSA. In other cases, SC-β cells can be obtained by culturing thepopulation of cells containing the insulin-positive endocrine cells in aMCDB131 medium that can be supplemented by 2% BSA. In some cases, theMCDB131 medium with 2% BSA for maturation of insulin-positive endocrinecells into SC-β cells can be comprise no small molecule factors asdescribed herein. In some case, the MCDB131 medium with 2% BSA formaturation of insulin-positive endocrine cells into SC-β cells cancomprise no serum (e.g., no FBS).

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingpluripotent stem cells in a population into definitive endoderm cells bycontacting the pluripotent stem cells with at least one factor from TGFβsuperfamily and a WNT signaling pathway activator for a period of 3days; b) differentiating at least some of the definitive endoderm cellsinto primitive gut tube cells by a process of contacting the definitiveendoderm cells with at least one factor from the FGF family for a periodof 3 days; c) differentiating at least some of the primitive gut tubecells into Pdx1-positive pancreatic progenitor cells by a process ofcontacting the primitive gut tube cells with i) retinoic acid signalingpathway activator, ii) at least one factor from the FGF family, iii) aSHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g.,DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor; d)differentiating at least some of the Pdx1-positive pancreatic progenitorcells into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells bya process of contacting the Pdx1-positive pancreatic progenitor cellsunder conditions that promote cell clustering with i) at least onegrowth factor from the FGF family, ii) at least one SHH pathwayinhibitor, and optionally iii) a RA signaling pathway activator, andoptionally iv) ROCK inhibitor and v) at least one factor from TGFβsuperfamily, every other day for a period of 5 days, wherein theNKX6.1-positive pancreatic progenitor cells expresses Pdx1 and NKX6.1;e) differentiating at least some of the Pdx1-positive, NKX6.1-positivepancreatic progenitor cells into Pdx1-positive, NKX6.1-positive,insulin-positive endocrine cells by a process of contacting thePdx1-positive, NKX6.1-positive pancreatic progenitor cells with i) aTGF-β signaling pathway inhibitor, ii) a TH signaling pathway activator,iii) at least one SHH pathway inhibitor, iv) a RA signaling pathwayactivator, v) a γ-secretase inhibitor, optionally vi) at least onegrowth factor from the epidermal growth factor (EGF) family, andoptionally vii) a BMP signaling pathway inhibitor, every other day for aperiod of between five and seven days; and f) differentiating at leastsome of the Pdx1-positive, NKX6.1-positive, insulin-positive endocrinecells into SC-β cells by a process of culturing the Pdx1-positive,NKX6.1-positive, insulin-positive endocrine cells in a medium (e.g.,NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, orDMEM/F12 medium) without exogenous differentiation factors, every otherday for a period of between 7 and 14 days to induce the in vitromaturation of at least some of the Pdx1-positive, NKX6.1-positive,insulin-positive endocrine cells into SC-β cells, wherein the SC-β cellsexhibit a GSIS response in vitro and/or in vivo. In some cases, the GSISresponse resembles the GSIS response of an endogenous mature β cells.

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingpluripotent stem cells in a population into definitive endoderm cells bycontacting the pluripotent stem cells with at least one factor from TGFβsuperfamily and a WNT signaling pathway activator for a period of 3days; b) differentiating at least some of the definitive endoderm cellsinto primitive gut tube cells by a process of contacting the definitiveendoderm cells with at least one factor from the FGF family for a periodof 3 days; c) differentiating at least some of the primitive gut tubecells into Pdx1-positive pancreatic progenitor cells by a process ofcontacting the primitive gut tube cells with i) retinoic acid signalingpathway activator, ii) at least one factor from the FGF family, iii) aSHH pathway inhibitor, iv) a BMP signaling pathway inhibitor, v) a PKCactivator, vi) a ROCK inhibitor, and vii) a growth factor from TGFβsuperfamily, for a period of 2 days; d) differentiating at least some ofthe Pdx1-positive pancreatic progenitor cells into Pdx1-positive,NKX6.1-positive pancreatic progenitor cells by a process of contactingthe Pdx1-positive pancreatic progenitor cells under conditions thatpromote cell clustering with i) at least one growth factor from the FGFfamily, ii) at least one SHH pathway inhibitor, and optionally iii) a RAsignaling pathway activator, and optionally iv) ROCK inhibitor and v) atleast one factor from TGFβ superfamily, every other day for a period of5 days, wherein the NKX6.1-positive pancreatic progenitor cellsexpresses Pdx1 and NKX6.1; e) differentiating at least some of thePdx1-positive, NKX6.1-positive pancreatic progenitor cells intoPdx1-positive, NKX6.1-positive, insulin-positive endocrine cells by aprocess of contacting the Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells with i) a TGF-β signaling pathway inhibitor, ii) a THsignaling pathway activator, iii) at least one SHH pathway inhibitor,iv) a RA signaling pathway activator, v) a γ-secretase inhibitor,optionally vi) at least one growth factor from the epidermal growthfactor (EGF) family, and optionally vii) a BMP signaling pathwayinhibitor, every other day for a period of between five and seven days;and f) differentiating at least some of the Pdx1-positive,NKX6.1-positive, insulin-positive endocrine cells into SC-β cells by aprocess of culturing the Pdx1-positive, NKX6.1-positive,insulin-positive endocrine cells in a medium (e.g., NS-GFs medium, MCDBmedium supplemented with BSA, MCDB131 medium, or DMEM/F12 medium)without exogenous differentiation factors, every other day for a periodof between 7 and 14 days to induce the in vitro maturation of at leastsome of the Pdx1-positive, NKX6.1-positive, insulin-positive endocrinecells into SC-β cells, wherein the SC-β cells exhibit a GSIS response invitro and/or in vivo. In some cases, the GSIS response resembles theGSIS response of an endogenous mature β cells.

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingpluripotent stem cells in a population into definitive endoderm cells bycontacting the pluripotent stem cells with at least one factor from TGFβsuperfamily and a WNT signaling pathway activator for a period of 3days; b) differentiating at least some of the definitive endoderm cellsinto primitive gut tube cells by a process of contacting the definitiveendoderm cells with at least one factor from the FGF family for a periodof 3 days; c) differentiating at least some of the primitive gut tubecells into Pdx1-positive pancreatic progenitor cells by a process ofcontacting the primitive gut tube cells with i) retinoic acid signalingpathway activator, ii) at least one factor from the FGF family, iii) aSHH pathway inhibitor, iv) a PKC activator, and v) a ROCK inhibitor; d)differentiating at least some of the Pdx1-positive pancreatic progenitorcells into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells bya process of contacting the Pdx1-positive pancreatic progenitor cellsunder conditions that promote cell clustering with i) at least onegrowth factor from the FGF family, ii) at least one SHH pathwayinhibitor, and optionally iii) a RA signaling pathway activator, andoptionally iv) ROCK inhibitor and v) at least one factor from TGFβsuperfamily, every other day for a period of 5 days, wherein theNKX6.1-positive pancreatic progenitor cells expresses Pdx1 and NKX6.1;e) differentiating at least some of the Pdx1-positive, NKX6.1-positivepancreatic progenitor cells into Pdx1-positive, NKX6.1-positive,insulin-positive endocrine cells by a process of contacting thePdx1-positive, NKX6.1-positive pancreatic progenitor cells with i) aTGF-β signaling pathway inhibitor, ii) a TH signaling pathway activator,iii) at least one SHH pathway inhibitor, iv) a RA signaling pathwayactivator, v) a γ-secretase inhibitor, and optionally vi) at least onegrowth factor from the epidermal growth factor (EGF) family, every otherday for a period of between five and seven days; and f) differentiatingat least some of the Pdx1-positive, NKX6.1-positive, insulin-positiveendocrine cells into SC-β cells by a process of culturing thePdx1-positive, NKX6.1-positive, insulin-positive endocrine cells in amedium (e.g., NS-GFs medium, MCDB medium supplemented with BSA, MCDB131medium, or DMEM/F12 medium) without exogenous differentiation factors,every other day for a period of between 7 and 14 days to induce the invitro maturation of at least some of the Pdx1-positive, NKX6.1-positive,insulin-positive endocrine cells into SC-β cells, wherein the SC-β cellsexhibit a GSIS response in vitro and/or in vivo. In some cases, the GSISresponse resembles the GSIS response of an endogenous mature β cells.

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingpluripotent stem cells in a population into definitive endoderm cells bycontacting the pluripotent stem cells with at least one factor from TGFβsuperfamily and a WNT signaling pathway activator for a period of 3days; b) differentiating at least some of the definitive endoderm cellsinto primitive gut tube cells by a process of contacting the definitiveendoderm cells with at least one factor from the FGF family for a periodof 3 days; c) differentiating at least some of the primitive gut tubecells into Pdx1-positive pancreatic progenitor cells by a process ofcontacting the primitive gut tube cells with i) retinoic acid signalingpathway activator, ii) at least one factor from the FGF family, iii) aSHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g.,DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor; d)differentiating at least some of the Pdx1-positive pancreatic progenitorcells into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells bya process of contacting the Pdx1-positive pancreatic progenitor cellsunder conditions that promote cell clustering with i) at least onegrowth factor from the FGF family, ii) at least one SHH pathwayinhibitor, and optionally iii) a RA signaling pathway activator, andoptionally iv) ROCK inhibitor and v) at least one factor from TGFβsuperfamily, every other day for a period of 5 or 6 days, wherein theNKX6.1-positive pancreatic progenitor cells expresses Pdx1 and NKX6.1;e) differentiating at least some of the Pdx1-positive, NKX6.1-positivepancreatic progenitor cells into Pdx1-positive, NKX6.1-positive,insulin-positive endocrine cells by a process of contacting thePdx1-positive, NKX6.1-positive pancreatic progenitor cells with i) a SHHpathway inhibitor, ii) a RA signaling pathway activator, iii) aγ-secretase inhibitor, iv) at least one growth factor from the epidermalgrowth factor (EGF) family, v) at least one bone morphogenetic protein(BMP) signaling pathway inhibitor, vi) a TGF-β signaling pathwayinhibitor, vii) a thyroid hormone signaling pathway activator, viii) anepigenetic modifying compound (e.g., DZNep or KD5170), ix) a proteinkinase inhibitor, and x) a ROCK inhibitor, every other day for a periodof between five and seven days; and f) differentiating at least some ofthe Pdx1-positive, NKX6.1-positive, insulin-positive endocrine cellsinto SC-β cells by a process of culturing the Pdx1-positive,NKX6.1-positive, insulin-positive endocrine cells in a medium (e.g.,NS-GFs medium, MCDB medium supplemented with BSA, MCDB131 medium, orDMEM/F12 medium) without exogenous differentiation factors, every otherday for a period of between 7 and 14 days to induce the in vitromaturation of at least some of the Pdx1-positive, NKX6.1-positive,insulin-positive endocrine cells into SC-β cells, wherein the SC-β cellsexhibit a GSIS response in vitro and/or in vivo. In some cases, the GSISresponse resembles the GSIS response of an endogenous mature β cells.

In some aspects, the disclosure provides a method of generating SC-βcells from pluripotent cells, the method comprising: a) differentiatingpluripotent stem cells in a population into definitive endoderm cells bycontacting the pluripotent stem cells with at least one factor from TGFβsuperfamily and a WNT signaling pathway activator for a period of 3days; b) differentiating at least some of the definitive endoderm cellsinto primitive gut tube cells by a process of contacting the definitiveendoderm cells with at least one factor from the FGF family for a periodof 3 days; c) differentiating at least some of the primitive gut tubecells into Pdx1-positive pancreatic progenitor cells by a process ofcontacting the primitive gut tube cells with i) retinoic acid signalingpathway activator, ii) at least one factor from the FGF family, iii) aSHH pathway inhibitor, iv) a BMP signaling pathway inhibitor (e.g.,DMH-1 or LDN193189), v) a PKC activator, and vi) a ROCK inhibitor; d)differentiating at least some of the Pdx1-positive pancreatic progenitorcells into Pdx1-positive, NKX6.1-positive pancreatic progenitor cells bya process of contacting the Pdx1-positive pancreatic progenitor cellsunder conditions that promote cell clustering with i) at least onegrowth factor from the FGF family, ii) at least one SHH pathwayinhibitor, and optionally iii) a RA signaling pathway activator, andoptionally iv) ROCK inhibitor and v) at least one factor from TGFβsuperfamily, every other day for a period of 5 or 6 days, wherein theNKX6.1-positive pancreatic progenitor cells expresses Pdx1 and NKX6.1;e) differentiating at least some of the Pdx1-positive, NKX6.1-positivepancreatic progenitor cells into Pdx1-positive, NKX6.1-positive,insulin-positive endocrine cells by a process of contacting thePdx1-positive, NKX6.1-positive pancreatic progenitor cells with i) aγ-secretase inhibitor, ii) at least one bone morphogenetic protein (BMP)signaling pathway inhibitor, iii) a TGF-β signaling pathway inhibitor,iv) a thyroid hormone signaling pathway activator, v) an epigeneticmodifying compound (e.g., DZNep or KD5170), vi) a protein kinaseinhibitor, and vii) a ROCK inhibitor, every other day for a period ofbetween five and seven days, and within first three days of the periodof between five and seven days, contacting the Pdx1-positive,NKX6.1-positive pancreatic progenitor cells with a SHH pathwayinhibitor, a RA signaling pathway, and at least one growth factor fromthe EGF family, which are removed from the Pdx1-positive,NKX6.1-positive pancreatic progenitor cells thereafter; and f)differentiating at least some of the Pdx1-positive, NKX6.1-positive,insulin-positive endocrine cells into SC-β cells by a process ofculturing the Pdx1-positive, NKX6.1-positive, insulin-positive endocrinecells in a medium (e.g., NS-GFs medium, MCDB medium supplemented withBSA, MCDB131 medium, or DMEM/F12 medium) without exogenousdifferentiation factors, every other day for a period of between 7 and14 days to induce the in vitro maturation of at least some of thePdx1-positive, NKX6.1-positive, insulin-positive endocrine cells intoSC-β cells, wherein the SC-β cells exhibit a GSIS response in vitroand/or in vivo. In some cases, the GSIS response resembles the GSISresponse of an endogenous mature β cells.

The medium used to culture the cells dissociated from the first cellcluster can be xeno-free. A xeno-free medium for culturing cells and/orcell clusters of originated from an animal can have no product fromother animals. In some cases, a xeno-free medium for culturing humancells and/or cell clusters can have no products from any non-humananimals. For example, a xeno-free medium for culturing human cellsand/or cell clusters can comprise human platelet lysate (PLT) instead offetal bovine serum (FBS). For example, a medium can comprise from about1% to about 20%, from about 5% to about 15%, from about 8% to about 12%,from about 9 to about 11% serum. In some cases, medium can compriseabout 10% of serum. In some cases, the medium can be free of smallmolecules and/or FBS. For example, a medium can comprise MCDB131 basalmedium supplemented with 2% BSA. In some cases, the medium isserum-free. In some examples, a medium can comprise no exogenous smallmolecules or signaling pathway agonists or antagonists, such as, growthfactor from fibroblast growth factor family (FGF, such as FGF2, FGF8B,FGF 10, or FGF21), Sonic Hedgehog Antagonist (such as Sant1, Sant2,Sant4, Sant4, Cur61414, forskolin, tomatidine, AY9944, triparanol,cyclopamine, or derivatives thereof), Retinoic Acid Signaling agonist(e.g., retinoic acid, CD1530, AM580, TTHPB, CD437, Ch55, BMS961,AC261066, AC55649, AM80, BMS753, tazarotene, adapalene, or CD2314),inhibitor of Rho-associated, coiled-coil containing protein kinase(ROCK) (e.g., Thiazovivin, Y-27632, Fasudil/HA1077, or 14-1152),activator of protein kinase C (PKC) (e.g., phorbol 12,13-dibutyrate(PDBU), TPB, phorbol 12-myristate 13-acetate, bryostatin 1, orderivatives thereof), antagonist of TGF β super family (e.g., Alk5inhibitor II (CAS 446859-33-2), A83-01, SB431542, D4476, GW788388,LY364947, LY580276, SB505124, GW6604, SB-525334, SD-208, SB-505124, orderivatives thereof), inhibitor of Bone Morphogenetic Protein (BMP) type1 receptor (e.g., LDN193189 or derivatives thereof), thyroid hormonesignaling pathway activator (e.g., T3, GC-1 or derivatives thereof),gamma-secretase inhibitor (e.g., XXI, DAPT, or derivatives thereof),activator of TGF-β signaling pathway (e.g., WNT3a or Activin A) growthfactor from epidermal growth factor (EGF) family (e.g., betacellulin orEGF), broad kinase (e.g., staurosporine or derivatives thereof),non-essential amino acids, vitamins or antioxidants (e.g., cyclopamine,vitamin D, vitamin C, vitamin A, or derivatives thereof), or otheradditions like N-acetyl cysteine, zinc sulfate, or heparin. In somecases, the reaggregation medium can comprise no exogenous extracellularmatrix molecule. In some cases, the reaggregation medium does notcomprise Matrigel™. In some cases, the reaggregation medium does notcomprise other extracellular matrix molecules or materials, such as,collagen, gelatin, poly-L-lysine, poly-D-lysine, vitronectin, laminin,fibronectin, PLO laminin, fibrin, thrombin, and RetroNectin and mixturesthereof, for example, or lysed cell membrane preparations.

A person of ordinary skill in the art will appreciate that that theconcentration of serum albumin supplemented into the medium may vary.For example, a medium (e.g., MCDB131) can comprise about 0.01%, 0.05%,0.1%, 1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about15% BSA. In other cases, a medium can comprise about 0.01%, 0.05%, 0.1%,1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about 15% HSA.The medium used (e.g., MCDB131 medium) can contain components not foundin traditional basal media, such as trace elements, putrescine, adenine,thymidine, and higher levels of some amino acids and vitamins. Theseadditions can allow the medium to be supplemented with very low levelsof serum or defined components. The medium can be free of proteinsand/or growth factors, and may be supplemented with EGF, hydrocortisone,and/or glutamine. The medium can comprise one or more extracellularmatrix molecules (e.g., extracellular proteins). Non-limiting exemplaryextracellular matrix molecules used in the medium can include collagen,placental matrix, fibronectin, laminin, merosin, tenascin, heparin,heparin sulfate, chondroitin sulfate, dermatan sulfate, aggrecan,biglycan, thrombospondin, vitronectin, and decorin. In some cases, themedium comprises laminin, such as LN-332. In some cases, the mediumcomprises heparin.

The medium can be changed periodically in the culture, e.g., to provideoptimal environment for the cells in the medium. When culturing thecells dissociated from the first cell cluster for re-aggregation, themedium can be changed at least or about every 4 hours, 12 hours, 24hours, 48 hours, 3 days or 4 days. For example, the medium can bechanged about every 48 hours.

In some cases, cells can be cultured under dynamic conditions (e.g.,under conditions in which the cells are subject to constant movement orstirring while in the suspension culture). For dynamic culturing ofcells, the cells can be cultured in a container (e.g., an non-adhesivecontainer such as a spinner flask (e.g., of 200 ml to 3000 ml, forexample 250 ml; of 100 ml; or in 125 ml Erlenmeyer), which can beconnected to a control unit and thus present a controlled culturingsystem. In some cases, cells can be cultured under non-dynamicconditions (e.g., a static culture) while preserving their proliferativecapacity. For non-dynamic culturing of cells, the cells can be culturedin an adherent culture vessel. An adhesive culture vessel can be coatedwith any of substrates for cell adhesion such as extracellular matrix(ECM) to improve the adhesiveness of the vessel surface to the cells.The substrate for cell adhesion can be any material intended to attachstem cells or feeder cells (if used). The substrate for cell adhesionincludes collagen, gelatin, poly-L-lysine, poly-D-lysine, vitronectin,laminin, fibronectin, PLO laminin, fibrin, thrombin, and RetroNectin andmixtures thereof, for example, Matrigel™, and lysed cell membranepreparations.

Medium in a dynamic cell culture vessel (e.g., a spinner flask) can bestirred (e.g., by a stirrer). The spinning speed can correlate with thesize of the re-aggregated second cell cluster. The spinning speed can becontrolled so that the size of the second cell cluster can be similar toan endogenous pancreatic islet. In some cases, the spinning speed iscontrolled so that the size of the second cell cluster can be from about75 μm to about 250 μm. The spinning speed of a dynamic cell culturevessel (e.g., a spinner flask) can be about 20 rounds per minute (rpm)to about 100 rpm, e.g., from about 30 rpm to about 90 rpm, from about 40rpm to about 60 rpm, from about 45 rpm to about 50 rpm. In some cases,the spinning speed can be about 50 rpm.

Stage 6 cells as provided herein may or may not be subject to thedissociation and reaggregation process as described herein. In somecases, the cell cluster comprising the insulin-positive endocrine cellscan be reaggregated. The reaggregation of the cell cluster can enrichthe insulin-positive endocrine cells. In some cases, theinsulin-positive endocrine cells in the cell cluster can be furthermatured into pancreatic β cells. For example, after reaggregation, thesecond cell cluster can exhibit in vitro GSIS, resembling nativepancreatic islet. For example, after reaggregation, the second cellcluster can comprise non-native pancreatic β cell that exhibits in vitroGSIS. In some embodiments, the reaggregation process can be performedaccording to the disclosure of PCT application PCT/US2018/043179, whichis incorporated herein by reference in its entirety.

Stage 6 cells obtained according to methods provided herein can havehigh recovery yield after cryopreservation and reaggregation procedures.In some cases, stage 6 cells that are obtained in a differentiationprocess that involves treatment of a BMP signaling pathway inhibitor(e.g., DMH-1 or LDN) and a growth factor from TGF-β superfamily (e.g.,Activin A) at stage 3 and treatment of an epigenetic modifying compound(e.g., histone methyltransferase inhibitor, e.g., EZH2 inhibitor, e.g.,DZNep) at stage 5 can have a higher recovery yield aftercryopreservation post stage 5, as compared to a corresponding cellpopulation without such treatment. In some cases, stage 6 cells that areobtained in a differentiation process that involves treatment of a BMPsignaling pathway inhibitor (e.g., DMH-1 or LDN) and a growth factorfrom TGF-β superfamily (e.g., Activin A) at stage 3 and treatment of anepigenetic modifying compound (e.g., histone methyltransferaseinhibitor, e.g., EZH2 inhibitor, e.g., DZNep) at stage 5 can have ahigher recovery yield after cryopreservation post stage 5, as comparedto a corresponding cell population without treatment of a BMP signalingpathway inhibitor (e.g., DMH-1 or LDN) and a growth factor from TGF-βsuperfamily (e.g., Activin A) at stage 3. In some cases, stage 6 cellsthat are obtained in a differentiation process that involves treatmentof a BMP signaling pathway inhibitor (e.g., DMH-1 or LDN) and a growthfactor from TGF-β superfamily (e.g., Activin A) at stage 3 and treatmentof an epigenetic modifying compound (e.g., histone methyltransferaseinhibitor, e.g., EZH2 inhibitor, e.g., DZNep) at stage 5 can have arecovery yield after cryopreservation post stage 5 that is at leastabout 35%, 37.5%, 40%, 42.5%, 45%, 47.5%, 48%, 49%, or 50%. The recoveryyield can be calculated as a percentage of cells that survive and formreaggregated cell clusters after cryopreservation, thawing and recovery,and reaggregation procedures, as compared to the cells before thecryopreservation.

In some embodiments, the present disclosure relates to cryopreservationof the non-native pancreatic β cells or precursors thereof obtainedusing the methods provided herein. In some embodiments, the cellpopulation comprising non-native pancreatic β cells can be stored viacryopreservation. For instances, the cell population comprisingnon-native β cells, e.g., Stage 6 cells in some cases, can bedissociated into cell suspension, e.g., single cell suspension, and thecell suspension can be cryopreserved, e.g., frozen in a cryopreservationsolution. The dissociation of the cells can be conducted by any of thetechnique provided herein, for example, by enzymatic treatment. Thecells can be frozen at a temperature of at highest −20° C., at highest−30° C., at highest −40° C., at highest −50° C., at highest −60° C., athighest −70° C., at highest −80° C., at highest −90° C., at highest−100° C., at highest −110° C., at highest −120° C., at highest −130° C.,at highest −140° C., at highest −150° C., at highest −160° C., athighest −170° C., at highest −180° C., at highest −190° C., or athighest −200° C. In some cases, the cells are frozen at a temperature ofabout −80° C. In some cases, the cells are frozen at a temperature ofabout −195° C. Any cooling methods can be used for providing the lowtemperature needed for cryopreservation, such as, but not limited to,electric freezer, solid carbon dioxide, and liquid nitrogen. In somecases, any cryopreservation solution available to one skilled in the artcan be used for incubating the cells for storage at low temperature,including both custom made and commercial solutions. For example, asolution containing a cryoprotectant can be used. The cryoprotectant canbe an agent that is configured to protect the cell from freezing damage.For instance, a cryoprotectant can be a substance that can lower theglass transition temperature of the cryopreservation solution. Exemplarycryoprotectants that can be used include DMSO (dimethyl sulfoxide),glycols (e.g., ethylene glycol, propylene glycol and glycerol), dextran(e.g., dextran-40), and trehalose. Additional agents can be added in tothe cryopreservation solution for other effects. In some cases,commercially available cryopreservation solutions can be used in themethod provided herein, for instance, FrostaLife™, pZerve™, PrimeXV®,Gibco Synth-a-Freeze Cryopreservation Medium, STEM-CELLBANKER®,CryoStor® Freezing Media, HypoThermosol® FRS Preservation Media, andCryoDefend® Stem Cells Media.

During the differentiation process, the cells can be subject toirradiation treatment as provided herein. In some cases, the cellpopulation at Stage 6, e.g., the cell population or cell cluster thathas cells being differentiated from insulin-positive endocrine cellsinto pancreatic β cells, is irradiated for a period of time. In somecases, the cell population at Stage 6 after reaggregation following therecovery from cryopreservation is irradiated for a period of time. Insome cases, the cryopreserved cells (e.g., the cells that arecryopreserved at the end of Stage 5) are irradiated for a certain periodof time prior to thawing and recovery for subsequent differentiationprocess.

V. Differentiation Factors

Aspects of the disclosure relate to contacting progenitor cells (e.g.,stem cells, e.g., iPS cells, definitive endoderm cells, primitive guttube cells, Pdx1-positive pancreatic progenitor cells, NKX6.1-positivepancreatic progenitor cells, insulin-positive endocrine cells) with βcell differentiation factors, for example, to induce the maturation ofthe insulin-positive endocrine cells or differentiation of otherprogenitor cells into SC-β cells (e.g., mature pancreatic β cells). Insome embodiments, the differentiation factor can induce thedifferentiation of pluripotent cells (e.g., iPSCs or hESCs) intodefinitive endoderm cells, e.g., in accordance with a method describedherein. In some embodiments, the differentiation factor can induce thedifferentiation of definitive endoderm cells into primitive gut tubecells, e.g., in accordance with a method described herein. In someembodiments, the differentiation factor can induce the differentiationof primitive gut tube cells into Pdx1-positive pancreatic progenitorcells, e.g., in accordance with a method described herein. In someembodiments, the differentiation factor can induce the differentiationof Pdx1-positive pancreatic progenitor cells into NKX6-1-positivepancreatic progenitor cells, e.g., in accordance with a method describedherein. In some embodiments, the differentiation factor can induce thedifferentiation of NKX6-1-positive pancreatic progenitor cells intoinsulin-positive endocrine cells, e.g., in accordance with a methoddescribed herein. In some embodiments, the differentiation factor caninduce the maturation of insulin-positive endocrine cells into SC-βcells, e.g., in accordance with a method described herein.

At least one differentiation factor described herein can be used alone,or in combination with other differentiation actors, to generate SC-βcells according to the methods as disclosed herein. In some embodiments,at least two, at least three, at least four, at least five, at leastsix, at least seven, at least eight, at least nine, or at least tendifferentiation factors described herein are used in the methods ofgenerating SC-β cells.

Transforming Growth Factor-β (TGF-β) Superfamily

Aspects of the disclosure relate to the use of growth factors from thetransforming growth factor-β (TGF-β) superfamily as differentiationfactors. The “TGF-β superfamily” means proteins having structural andfunctional characteristics of known TGFβ family members. The TGFβ familyof proteins can include the TGFβ series of proteins, the Inhibins(including Inhibin A and Inhibin B), the Activins (including Activin A,Activin B, and Activin AB), MIS (Müllerian inhibiting substance), BMP(bone morphogenetic proteins), dpp (decapentaplegic), Vg-1, MNSF(monoclonal nonspecific suppressor factor), and others. Activity of thisfamily of proteins can be based on specific binding to certain receptorson various cell types. Members of this family can share regions ofsequence identity, particularly at the C-terminus, that correlate totheir function. The TGFβ family can include more than one hundreddistinct proteins, all sharing at least one region of amino acidsequence identity. Members of the family that can be used in the methoddisclosed herein can include, but are not limited to, the followingproteins, as identified by their GenBank accession numbers: P07995,P18331, P08476, Q04998, P03970, P43032, P55102, P27092, P42917, P09529,P27093, P04088, Q04999, P17491, P55104, Q9WUK5, P55103, O88959, O08717,P58166, O61643, P35621, P09534, P48970, Q9NR23, P25703, P30884, P12643,P49001, P21274, O46564, O19006, P22004, P20722, Q04906, Q07104, P30886,P18075, P23359, P22003, P34821, P49003, Q90751, P21275, Q06826, P30885,P34820, Q29607, P12644, Q90752, 046576, P27539, P48969, Q26974, P07713,P91706, P91699, P27091, 042222, Q24735, P20863, O18828, P55106, Q9PTQ2,O14793, O08689, O42221, O18830, O18831, O18836, O35312, O42220, P43026,P43027, P43029, O95390, Q9R229, O93449, Q9Z1W4, Q9BDW8, P43028, Q7Z4P5,P50414, P17246, P54831, P04202, P01137, P09533, P18341, O19011, Q9Z1Y6,P07200, Q9Z217, O95393, P55105, P30371, Q9MZE2, Q07258, Q96S42, P97737,AAA97415.1, NP-776788.1, NP-058824.1, EAL24001.1, 1 S4Y, NP-001009856.1,NP-1-032406.1, NP-999193.1, XP-519063.1, AAG17260.1, CAA40806.1,NP-1-001009458.1, AAQ55808.1, AAK40341.1, AAP33019.1, AAK21265.1,AAC59738.1, CAI46003.1, B40905, AAQ55811.1, AAK40342.1, XP-540364.1,P55102, AAQ55810.1, NP-990727.1, CAA51163.1, AAD50448.1, JC4862, PN0504,BAB17600.1, AAH56742.1, BAB17596.1, CAG06183.1, CAG05339.1, BAB17601.1,CAB43091.1, A36192, AAA49162.1, AAT42200.1, NP-789822.1, AAA59451.1,AAA59169.1, XP-541000.1, NP-990537.1, NP-1-002184.1, AAC14187.1,AAP83319.1, AAA59170.1, BAB16973.1, AAM66766.1, WFPGBB, 1201278C,AAH30029.1, CAA49326.1, XP-344131.1, AA-148845.1, XP-1-148966.3, 148235,B41398, AAH77857.1, AAB26863.1, 1706327A, BAA83804.1, NP-571143.1,CAG00858.1, BAB17599.1, BAB17602.1, AAB61468.1, PN0505, PN0506,CAB43092.1, BAB17598.1, BAA22570.1, BAB16972.1, BAC81672.1, BAA12694.1,BAA08494.1, B36192, C36192, BAB16971.1, NP-034695.1, AAA49160.1,CAA62347.1, AAA49161.1, AAD30132.1, CAA58290.1, NP-005529.1,XP-522443.1, AAM27448.1, XP-538247.1, AAD30133.I, AAC36741.1,AAH10404.1, NP-032408.1, AAN03682.1, XP-509161.1, AAC32311.1,NP-651942.2, AAL51005.1, AAC39083.1, AAH85547.1, NP-571023.1,CAF94113.1, EAL29247.1, AAW30007.1, AAH90232.1, A29619, NP-001007905.1,AAH73508.1, AAD02201.1, NP-999793.1, NP-990542.1, AAF19841.1,AAC97488.1, AAC60038.1, NP 989197.1, NP-571434.1, EAL41229.1,AAT07302.1, CAI19472.1, NP-031582.1, AAA40548.1, XP-535880.1,NP-1-037239.1, AAT72007.1, XP-418956.1, CAA41634.1, BAC30864.1,CAA38850.1, CAB81657.2, CAA45018.1, CAA45019.1, BAC28247.1, NP-031581.1,NP-990479.1, NP-999820.1, AAB27335.1, 545355, CAB82007.1, XP-534351.1,NP-058874.1, NP-031579.1, 1REW, AAB96785.1, AAB46367.1, CAA05033.1,BAA89012.1, IES7, AAP20870.1, BAC24087.1, AAG09784.1, BAC06352.1,AAQ89234.1, AAM27000.1, AAH30959.1, CAGO1491.1, NP-571435.1, 1REU,AAC60286.1, BAA24406.1, A36193, AAH55959.1, AAH54647.1, AAH90689.1,CAG09422.1, BAD16743.1, NP-032134.1, XP-532179.1, AAB24876.1,AAH57702.1, AAA82616.1, CAA40222.1, CAB90273.2, XP-342592.1,XP-534896.1, XP-534462.1, 1LXI, XP-417496.1, AAF34179.1, AAL73188.1,CAF96266.1, AAB34226.1, AAB33846.1, AAT12415.1, AA033819.1, AAT72008.1,AAD38402.1, BAB68396.1, CAA45021.1, AAB27337.1, AAP69917.1, AATI2416.1,NP-571396.1, CAA53513.1, AA033820.1, AAA48568.1, BAC02605.1, BAC02604.1,BAC02603.1, BAC02602.1, BAC02601.1, BAC02599.1, BAC02598.1, BAC02597.1,BAC02595.1, BAC02593.1, BAC02592.1, BAC02590.1, AAD28039.1, AAP74560.1,AAB94786.1, NP-001483.2, XP-528195.1, NP-571417.1, NP-001001557.I,AAH43222.1, AAM33143.1, CAG10381.1, BAA31132.1, EAL39680.1, EAA12482.2,P34820, AAP88972.1, AAP74559.1, CAI16418.1, AAD30538.1, XP-345502.1,NP-1-038554.1, CAG04089.1, CAD60936.2, NP-031584.1, B55452, AAC60285.1,BAA06410.1, AAH52846.1, NP-031580.1, NP-1-036959.1, CAA45836.1,CAA45020.1, Q29607, AAB27336.1, XP-547817.1, AAT12414.1, AAM54049.1,AAH78901.1, AA025745.1, NP-570912.1, XP-392194.1, AAD20829.1,AAC97113.1, AAC61694.1, AAH60340.1, AAR97906.1, BAA32227.1, BAB68395.1,BAC02895.1, AAWS 1451.1, AAF82188.1, XP-544189.1, NP-990568.1,BAC80211.1, AAW82620.1, AAF99597.1, NP-571062.1, CAC44179.1, AAB97467.1,AAT99303.1, AAD28038.1, AAH52168.1, NP-001004122.1, CAA72733.1,NP-032133.2, XP-394252.1, XP-224733.2, JH0801, AAP97721.1, NP-989669.1,543296, P43029, A55452, AAH32495.1, XP-542974.1, NP-032135.1,AAK30842.1, AAK27794.1, BAC30847.1, EAA12064.2, AAP97720.1, XP-525704.1,AAT07301.1, BAD07014.1, CAF94356.1, AAR27581.1, AAG13400.1, AAC60127.1,CAF92055.1, XP-540103.1, AA020895.1, CAF97447.1, AAS01764.1, BAD08319.1,CAA10268.1, NP-998140.1, AAR03824.1, AAS48405.1, AAS48403.1, AAK53545.1,AAK84666.1, XP-395420.1, AAK56941.1, AAC47555.1, AAR88255.1, EAL33036.1,AAW47740.1, AAW29442.1, NP-722813.1, AAR08901.1, AAO 15420.2,CAC59700.1, AAL26886.1, AAK71708.1, AAK71707.1, CAC51427.2, AAK67984.1,AAK67983.1, AAK28706.1, P07713, P91706, P91699, CAG02450.1, AAC47552.1,NP-005802.1, XP-343149.1, AW34055.1, XP-538221.1, AAR27580.1,XP-125935.3, AAF21633.1, AAF21630.1, AAD05267.1, Q9Z1 W4, NP-1-031585.2,NP-571094.1, CAD43439.1, CAF99217.1, CAB63584.1, NP-722840.1,CAE46407.1, XP-1-417667.1, BAC53989.1, BAB19659.1, AAM46922.1,AAA81169.1, AAK28707.1, AAL05943.1, AAB17573.1, CAH25443.1, CAG10269.1,BAD16731.1, EAA00276.2, AAT07320.1, AAT07300.1, AAN15037.1, CAH25442.1,AAK08152.2, 2009388A, AAR12161.1, CAGO1961.1, CAB63656.1, CAD67714.1,CAF94162.1, NP-477340.1, EAL24792.1, NP-1-001009428.1, AAB86686.1,AAT40572.1, AAT40571.1, AAT40569.1, NP-033886.1, AAB49985.1, AAG39266.1,Q26974, AAC77461.1, AAC47262.1, BAC05509.1, NP-055297.1, XP-546146.1,XP-525772.1, NP-060525.2, AAH33585.1, AAH69080.1, CAG12751.1,AAH74757.2, NP-034964.1, NP-038639.1, 042221, AAF02773.1, NP-062024.1,AAR18244.1, AAR14343.1, XP-228285.2, AAT40573.1, AAT94456.1, AAL35278.1,AAL35277.1, AAL17640.1, AAC08035.1, AAB86692.1, CAB40844.1, BAC38637.1,BAB16046.1, AAN63522.1, NP-571041.1, AAB04986.2, AAC26791.1, AAB95254.1,BAA11835.1, AAR18246.1, XP-538528.1, BAA31853.1, AAK18000.1,XP-1-420540.1, AAL35276.1, AAQ98602.1, CAE71944.1, AAW50585.1,AAV63982.1, AAW29941.1, AAN87890.1, AAT40568.1, CAD57730.1, AAB81508.1,AAS00534.1, AAC59736.1, BAB79498.1, AAA97392.1, AAP85526.1, NP-999600.2,NP-878293.1, BAC82629.1, CAC60268.1, CAG04919.1, AAN10123.1, CAA07707.1AAK20912.1, AAR88254.1, CAC34629.1, AAL35275.1, AAD46997.I, AAN03842.1,NP-571951.2, CAC50881.1, AAL99367.1, AAL49502.1, AAB71839.1, AAB65415.1,NP-624359.1, NP-990153.1, AAF78069.1, AAK49790.1, NP-919367.2,NP-001192.1, XP-544948.1, AAQ18013.1, AAV38739.1, NP-851298.1,CAA67685.1, AAT67171.1, AAT37502.1, AAD27804.1, AAN76665.1, BAC11909.1,XP-1-421648.1, CAB63704.1, NP-037306.1, A55706, AAF02780.1, CAG09623.1,NP-067589.1, NP-035707.1, AAV30547.1, AAP49817.1, BAC77407.1,AAL87199.1, CAG07172.1, B36193, CAA33024.1, NP-1-001009400.1,AAP36538.1, XP-512687.1, XP-510080.1, AAH05513.1, 1KTZ, AAH14690.1,AAA31526.1.

The growth factor from the TGF-β superfamily in the methods andcompositions provided herein can be naturally obtained or recombinant.In some embodiments, the growth factor from the TGF-β superfamilycomprises Activin A. The term “Activin A” can include fragments andderivatives of Activin A. The sequence of an exemplary Activin A (SEQ IDNO: 1) is disclosed as SEQ ID NO: 1 in U.S. Pub. No. 2009/0155218 (the'218 publication). Other non-limiting examples of Activin A (SEQ ID NOS:2-16) are provided in SEQ ID NO: 2-16 of the '218 publication, andnon-limiting examples of nucleic acids encoding Activin A (SEQ ID NOS:17-18) are provided in SEQ ID NO:33-34 of the '218 publication. In someembodiments, the growth factor from the TGF-β superfamily can comprise apolypeptide having an amino acid sequence at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 99%, or greater identical to SEQ ID NO: 1 of the'218 publication (SEQ ID NO: 1).

In some embodiments, the growth factor from the TGF-β superfamilycomprises growth differentiation factor 8 (GDF8). The term “GDF8” caninclude fragments and derivatives of GDF8. The sequences of GDF8polypeptides are available to the skilled artisan. In some embodiments,the growth factor from the TGF-β superfamily comprises a polypeptidehaving an amino acid sequence at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, orat least 99%, or greater identical to the human GDF8 polypeptidesequence (GenBank Accession EAX10880).

In some embodiments, the growth factor from the TGF-β superfamilycomprises a growth factor that is closely related to GDF8, e.g., growthdifferentiation factor 11 (GDF11). In some embodiments, the growthfactor from the TGF-β superfamily comprises a polypeptide having anamino acid sequence at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least99%, or greater identical to the human GDF11 polypeptide sequence(GenBank Accession AAF21630).

In some embodiments, the growth factor from the TGF-β superfamily can bereplaced with an agent mimics the at least one growth factor from theTGF-β superfamily. Exemplary agents that mimic the at least one growthfactor from the TGF-β superfamily, include, without limitation, IDE1 andIDE2.

Bone Morphogenetic Protein (BMP) Signaling Pathway Inhibitors

Aspects of the disclosure relate to the use of BMP signaling pathwayinhibitors as β cell differentiation factors. The BMP signaling familyis a diverse subset of the TGF-β superfamily (Sebald et al. Biol. Chem.385:697-710, 2004). Over twenty known BMP ligands are recognized bythree distinct type II (BMPRII, ActRIIa, and ActRIIb) and at least threetype I (ALK2, ALK3, and ALK6) receptors. Dimeric ligands facilitateassembly of receptor heteromers, allowing the constitutively-active typeII receptor serine/threonine kinases to phosphorylate type I receptorserine/threonine kinases. Activated type I receptors phosphorylateBMP-responsive (BR—) SMAD effectors (SMADs 1, 5, and 8) to facilitatenuclear translocation in complex with SMAD4, a co-SMAD that alsofacilitates TGF signaling. In addition, BMP signals can activateintracellular effectors such as MAPK p38 in a SMAD-independent manner(Nohe et al. Cell Signal 16:291-299, 2004). Soluble BMP antagonists suchas noggin, chordin, gremlin, and follistatin limit BMP signaling byligand sequestration.

In some embodiments, the BMP signaling pathway inhibitor in the methodsand composition provided herein comprises DMH-1, or a derivative,analogue, or variant thereof. In some embodiments, the BMP signalingpathway inhibitor in the methods and composition provided hereincomprises the following compound or a derivative, analogue, or variantof the following compound:

In some embodiments, the BMP signaling pathway inhibitor in the methodsand composition provided herein comprises LDN193189 (also known asLDN193189, 1062368-24-4, LDN-193189, DM 3189, DM-3189, IUPAC Name:4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinolone).In some embodiments, the BMP signaling pathway inhibitor in the methodsand composition provided herein comprises the following compound or aderivative, analogue, or variant of the following compound:

In some cases, DMH-1 can be more selective as compared to LDN193189. Insome embodiments of the present disclosure, DMH-1 can be particularlyuseful for the methods provided herein. In some embodiments, the methodsand compositions provided herein exclude use of LDN193189. In someembodiments, the methods and compositions provided herein exclude use ofLDN193189, or a derivative, analogue, or variant thereof for generatingPdx1-positive pancreatic progenitor cells from primitive gut tube cells.In some embodiments, the methods and compositions provided herein relateto use of DMH-1, or a derivative, analogue, or variant thereof forgenerating Pdx1-positive pancreatic progenitor cells from primitive guttube cells.

In some embodiments, the BMP signaling pathway inhibitor in the methodsand composition provided herein comprise an analog or derivative ofLDN193189, e.g., a salt, hydrate, solvent, ester, or prodrug ofLDN193189. In some embodiments, a derivative (e.g., salt) of LDN193189comprises LDN193189 hydrochloride.

In some embodiments, the BMP signaling pathway inhibitor in the methodsand composition provided herein comprises a compound of Formula I fromU.S. Patent Publication No. 2011/0053930.

TGF-β Signaling Pathway Inhibitors

Aspects of the disclosure relate to the use of TGF-β signaling pathwayinhibitors as β cell differentiation factors.

In some embodiments, the TGF-β signaling pathway comprises TGF-βreceptor type I kinase (TGF-β RI) signaling. In some embodiments, theTGF-β signaling pathway inhibitor comprises ALK5 inhibitor II (CAS446859-33-2, an ATP-competitive inhibitor of TGF-B RI kinase, also knownas RepSox, IUPAC Name:2-[5-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine. In someembodiments, the TGF-β signaling pathway inhibitor is an analog orderivative of ALK5 inhibitor II.

In some embodiments, the analog or derivative of ALK5 inhibitor II (alsonamed “ALK5i”) is a compound of Formula I as described in U.S. PatentPublication No. 2012/0021519, incorporated by reference herein in itsentirety.

In some embodiments, the TGF-β signaling pathway inhibitor in themethods and compositions provided herein is a TGF-β receptor inhibitordescribed in U.S. Patent Publication No. 2010/0267731. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein comprises an ALK5 inhibitor described inU.S. Patent Publication Nos. 2009/0186076 and 2007/0142376. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is A 83-01. In some embodiments, the TGF-βsignaling pathway inhibitor in the methods and compositions providedherein is not A 83-01. In some embodiments, the compositions and methodsdescribed herein exclude A 83-01. In some embodiments, the TGF-βsignaling pathway inhibitor in the methods and compositions providedherein is SB 431542. In some embodiments, the TGF-β signaling pathwayinhibitor is not SB 431542. In some embodiments, the compositions andmethods described herein exclude SB 431542. In some embodiments, theTGF-β signaling pathway inhibitor in the methods and compositionsprovided herein is D 4476. In some embodiments, the TGF-β signalingpathway inhibitor is not D 4476. In some embodiments, the compositionsand methods described herein exclude D 4476. In some embodiments, theTGF-β signaling pathway inhibitor in the methods and compositionsprovided herein is GW 788388. In some embodiments, the TGF-β signalingpathway inhibitor is not GW 788388. In some embodiments, thecompositions and methods described herein exclude GW 788388. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is LY 364947. In some embodiments, theTGF-β signaling pathway inhibitor is not LY 364947. In some embodiments,the compositions and methods described herein exclude LY 364947. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is LY 580276. In some embodiments, theTGF-β signaling pathway inhibitor is not LY 580276. In some embodiments,the compositions and methods described herein exclude LY 580276. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is SB 525334. In some embodiments, theTGF-β signaling pathway inhibitor is not SB 525334. In some embodiments,the compositions and methods described herein exclude SB 525334. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is SB 505124. In some embodiments, theTGF-β signaling pathway inhibitor is not SB 505124. In some embodiments,the compositions and methods described herein exclude SB 505124. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is SD 208. In some embodiments, the TGF-βsignaling pathway inhibitor is not SD 208. In some embodiments, thecompositions and methods described herein exclude SD 208. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is GW 6604. In some embodiments, the TGF-βsignaling pathway inhibitor is not GW 6604. In some embodiments, thecompositions and methods described herein exclude GW 6604. In someembodiments, the TGF-β signaling pathway inhibitor in the methods andcompositions provided herein is GW 788388. In some embodiments, theTGF-β signaling pathway inhibitor in the methods and compositionsprovided herein is not GW 788388. In some embodiments, the compositionsand methods described herein exclude GW 788388.

From the collection of compounds described above, the following can beobtained from various sources: LY-364947, SB-525334, SD-208, andSB-505124 available from Sigma, P.O. Box 14508, St. Louis, Mo.,63178-9916; 616452 and 616453 available from Calbiochem (EMD Chemicals,Inc.), 480 S. Democrat Road, Gibbstown, N.J., 08027; GW788388 and GW6604available from GlaxoSmithKline, 980 Great West Road, Brentford,Middlesex, TW8 9GS, United Kingdom; LY580276 available from LillyResearch, Indianapolis, Ind. 46285; and SM16 available from Biogen Idec,P.O. Box 14627, 5000 Davis Drive, Research Triangle Park, N.C.,27709-4627.

WNT Signaling Pathway

Aspects of the disclosure relate to the use of activators of the WNTsignaling pathway as β cell differentiation factors.

In some embodiments, the WNT signaling pathway activator in the methodsand compositions provided herein comprises CHIR99021. In someembodiments, the WNT signaling pathway activator in the methods andcompositions provided herein comprises a derivative of CHIR99021, e.g.,a salt of CHIR99021, e.g., trihydrochloride, a hydrochloride salt ofCHIR99021. In some embodiments, the WNT signaling pathway activator inthe methods and compositions provided herein comprises Wnt3a recombinantprotein. In some embodiments, the WNT signaling pathway activator in themethods and compositions provided herein comprises a glycogen synthasekinase 3 (GSK3) inhibitor. Exemplary GSK3 inhibitors include, withoutlimitation, 3F8, A 1070722, AR-A 014418, BIO, BIO-acetoxime, FRATide,10Z-Hymenialdisine, Indirubin-3′oxime, kenpaullone, L803, L803-mts,lithium carbonate, NSC 693868, SB 216763, SB 415286, TC-G 24, TCS 2002,TCS 21311, TWS 119, and analogs or derivatives of any of these. Incertain embodiments, the methods, compositions, and kits disclosedherein exclude a WNT signaling pathway activator.

Fibroblast Growth Factor (FGF) Family

Aspects of the disclosure relate to the use of growth factors from theFGF family as β cell differentiation factors.

In some embodiments, the growth factor from the FGF family in themethods and compositions provided herein comprises keratinocyte growthfactor (KGF). The polypeptide sequences of KGF are available to theskilled artisan. In some embodiments, the growth factor from the FGFfamily comprises a polypeptide having an amino acid sequence at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or at least 99%, or greater identicalto the human KGF polypeptide sequence (GenBank Accession AAB21431).

In some embodiments, the growth factor from the FGF family in themethods and composition provided herein comprises FGF2. The polypeptidesequences of FGF2 are available to the skilled artisan. In someembodiments, the growth factor from the FGF family comprises apolypeptide having an amino acid sequence at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 99%, or greater identical to the human FGF2polypeptide sequence (GenBank Accession NP_001997).

In some embodiments, the at least one growth factor from the FGF familyin the methods and composition provided herein comprises FGF8B. Thepolypeptide sequences of FGF8B are available to the skilled artisan. Insome embodiments, the growth factor from the FGF family comprises apolypeptide having an amino acid sequence at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 99%, or greater identical to the human FGF8Bpolypeptide sequence (GenBank Accession AAB40954).

In some embodiments, the at least one growth factor from the FGF familyin the methods and composition provided herein comprises FGF10. Thepolypeptide sequences of FGF10 are available to the skilled artisan. Insome embodiments, the growth factor from the FGF family comprises apolypeptide having an amino acid sequence at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 99%, or greater identical to the human FGF10polypeptide sequence (GenBank Accession CAG46489).

In some embodiments, the at least one growth factor from the FGF familyin the methods and composition provided herein comprises FGF21. Thepolypeptide sequences of FGF21 are available to the skilled artisan. Insome embodiments, the growth factor from the FGF family comprises apolypeptide having an amino acid sequence at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 99%, or greater identical to the human FGF21polypeptide sequence (GenBank Accession AAQ89444.1).

Sonic Hedgehog (SHH) Signaling Pathway

Aspects of the disclosure relate to the use of SHH signaling pathwayinhibitors as 13 cell differentiation factors.

In some embodiments, the SHH signaling pathway inhibitor in the methodsand composition provided herein comprises Sant1. In some embodiments,the SHH signaling pathway inhibitor in the methods and compositionprovided herein comprises SANT2. In some embodiments, the SHH signalingpathway inhibitor in the methods and composition provided hereincomprises SANT3. In some embodiments, the SHH signaling pathwayinhibitor in the methods and composition provided herein comprisesSANT4. In some embodiments, the SI-1H signaling pathway inhibitorcomprises Cur61414. In some embodiments, the SHH signaling pathwayinhibitor in the methods and composition provided herein comprisesforskolin. In some embodiments, the SHH signaling pathway inhibitor inthe methods and composition provided herein comprises tomatidine. Insome embodiments, the SHH signaling pathway inhibitor in the methods andcomposition provided herein comprises AY9944. In some embodiments, theSHH signaling pathway inhibitor in the methods and composition providedherein comprises triparanol. In some embodiments, the SHH signalingpathway inhibitor in the methods and composition provided hereincomprises compound A or compound B (as disclosed in U.S. Pub. No.2004/0060568). In some embodiments, the SHH signaling pathway inhibitorin the methods and composition provided herein comprises a steroidalalkaloid that antagonizes hedgehog signaling (e.g., cyclopamine or aderivative thereof) as disclosed in U.S. Pub. No. 2006/0276391. Incertain embodiments, the methods, compositions, and kits disclosedherein exclude a SHH signaling pathway inhibitor.

Rho Kinase (ROCK) Signaling Pathway

Aspects of the disclosure relate to the use of ROCK signaling pathwayinhibitors (ROCK inhibitors) as β cell differentiation factors.

In some embodiments, the ROCK inhibitor in the methods and compositionprovided herein comprises Y-27632 or Thiazovivin. In some embodiments,the ROCK inhibitor in the methods and composition provided hereincomprises Thiazovivin. In some embodiments, the ROCK inhibitor in themethods and composition provided herein comprises Y-27632. In somecases, the ROCK inhibitor in the methods and composition provided hereincomprises the following compound or a derivative thereof:

In some cases, the ROCK inhibitor in the methods and compositionprovided herein comprises the following compound or a derivativethereof:

Non-limiting examples of ROCK inhibitor that can be used in the methodsand compositions provided herein include Thiazovivin, Y-27632,Fasudil/HA1077, H-1152, Ripasudil, Y39983, Wf-536, SLx-2119,Azabenzimidazole-aminofurazans, DE-104, Olefins, Isoquinolines,Indazoles, and pyridinealkene derivatives, ROKα inhibitor, XD-4000,HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamides,Rhostatin, BA-210, BA-207, BA-215, BA-285, BA-1037, Ki-23095, VAS-012,and quinazoline.

Retinoic Acid Signaling Pathway

Aspects of the disclosure relate to the use of modulators of retinoicacid signaling as 13 cell differentiation factors.

In some embodiments, the modulator of retinoic acid signaling in themethods and composition provided herein comprises an activator ofretinoic acid signaling. In some embodiments, the RA signaling pathwayactivator in the methods and composition provided herein comprisesretinoic acid. In some embodiments, the RA signaling pathway activatorin the methods and composition provided herein comprises a retinoic acidreceptor agonist. Exemplary retinoic acid receptor agonists in themethods and composition provided herein include, without limitation, CD1530, AM 580, TTNPB, CD 437, Ch 55, BMS 961, AC 261066, AC 55649, AM 80,BMS 753, tazarotene, adapalene, and CD 2314.

In some embodiments, the modulator of retinoic acid signaling in themethods and composition provided herein comprises an inhibitor ofretinoic acid signaling. In some embodiments, the retinoic acidsignaling pathway inhibitor comprises DEAB (IUPAC Name:2-[2-(diethylamino)ethoxy]-3-prop-2-enylbenzaldehyde). In someembodiments, the retinoic acid signaling pathway inhibitor comprises ananalog or derivative of DEAB.

In some embodiments, the retinoic acid signaling pathway inhibitor inthe methods and composition provided herein comprises a retinoic acidreceptor antagonist. In some embodiments, the retinoic acid receptorantagonist in the methods and composition provided herein comprises(E)-4-[2-(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethenyl]benzoicacid,(E)-4-[[(5,6-dihydro-5,5-dimethyl-8-phenylethynyl)-2-naphthalenyl]ethenyl]benzoicacid,(E)-4-[2-[5,6-dihydro-5,5-dimethyl-8-(2-naphthalenyl)-2-naphthalenyl]ethenyl]-benzoicacid, and(E)-4-[2-[5,6-dihydro-5,5-dimethyl-8-(4-methoxyphenyl)-2-naphthalenyl]ethenyl]benzoicacid. In some embodiments, the retinoic acid receptor antagonistcomprises BMS 195614 (CAS #253310-42-8), ER 50891 (CAS #187400-85-7),BMS 493 (CAS #170355-78-9), CD 2665 (CAS #170355-78-9), LE 135 (CAS#155877-83-1), BMS 453 (CAS #166977-43-1), or MM 11253 (CAS#345952-44-5).

In certain embodiments, the methods, compositions, and kits disclosedherein exclude a modulator of retinoic acid signaling. In certainembodiments, the methods, compositions, and kits disclosed hereinexclude a retinoic acid signaling pathway activator. In certainembodiments, the methods, compositions, and kits disclosed hereinexclude a retinoic acid signaling pathway inhibitor.

Protein Kinase C

Aspects of the disclosure relate to the use of protein kinase Cactivators as β cell differentiation factors. Protein kinase C is one ofthe largest families of protein kinase enzymes and is composed of avariety of isoforms. Conventional isoforms include a, βI, βII, γ; novelisoforms include δ, ε, η, Θ; and atypical isoforms include ξ and ι/λ PKCenzymes are primarily cytosolic but translocate to the membrane whenactivated. In the cytoplasm, PKC is phosphorylated by other kinases orautophosphorylates. In order to be activated, some PKC isoforms (e.g.,PKC-ε) require a molecule to bind to the diacylglycerol (“DAG”) bindingsite or the phosphatidylserine (“PS”) binding site. Others are able tobe activated without any secondary binding messengers at all. PKCactivators that bind to the DAG site include, but are not limited to,bryostatin, picologues, phorbol esters, aplysiatoxin, and gnidimacrin.PKC activators that bind to the PS site include, but are not limited to,polyunsaturated fatty acids and their derivatives. It is contemplatedthat any protein kinase C activator that is capable, either alone or incombination with one or more other β cell differentiation factors, ofinducing the differentiation of at least one insulin-producing,endocrine cell or precursor thereof into a SC-β cell can be used in themethods, compositions, and kits described herein.

In some embodiments, the PKC activator in the methods and compositionprovided herein comprises PdbU. In some embodiments, the PKC activatorin the methods and composition provided herein comprises TPB. In someembodiments, the PKC activator in the methods and composition providedherein comprises cyclopropanated polyunsaturated fatty acids,cyclopropanated monounsaturated fatty acids, cyclopropanatedpolyunsaturated fatty alcohols, cyclopropanated monounsaturated fattyalcohols, cyclopropanated polyunsaturated fatty acid esters,cyclopropanated monounsaturated fatty acid esters, cyclopropanatedpolyunsaturated fatty acid sulfates, cyclopropanated monounsaturatedfatty acid sulfates, cyclopropanated polyunsaturated fatty acidphosphates, cyclopropanated monounsaturated fatty acid phosphates,macrocyclic lactones, DAG derivatives, isoprenoids, octylindolactam V,gnidimacrin, iripallidal, ingenol, napthalenesulfonamides,diacylglycerol kinase inhibitors, fibroblast growth factor 18 (FGF-18),insulin growth factor, hormones, and growth factor activators, asdescribed in WIPO Pub. No. WO/2013/071282. In some embodiments, thebryostain comprises bryostatin-1, bryostatin-2, bryostatin-3,bryostatin-4, bryostatin-5, bryostatin-6, bryostatin-7, bryostatin-8,bryostatin-9, bryostatin-10, bryostatin-11, bryostatin-12,bryostatin-13, bryostatin-14, bryostatin-15, bryostatin-16,bryostatin-17, or bryostatin-18. In certain embodiments, the methods,compositions, and kits disclosed herein exclude a protein kinase Cactivator.

γ-Secretase Inhibitors

Aspects of the disclosure relate to the use of γ-secretase inhibitors asβ cell differentiation factors.

In some embodiments, the γ-secretase inhibitor in the methods andcomposition provided herein comprises XXI. In some embodiments, theγ-secretase inhibitor in the methods and composition provided hereincomprises DAPT. Additional exemplary γ-secretase inhibitors in themethods and composition provided herein include, without limitation, theγ-secretase inhibitors described in U.S. Pat. Nos. 7,049,296, 8,481,499,8,501,813, and WIPO Pub. No. WO/2013/052700. In certain embodiments, themethods, compositions, and kits disclosed herein exclude a γ-secretaseinhibitor.

Thyroid Hormone Signaling Pathway Activators

Aspects of the disclosure relate to the use of thyroid hormone signalingpathway activators as β cell differentiation factors.

In some embodiments, the thyroid hormone signaling pathway activator inthe methods and composition provided herein comprises triiodothyronine(T3). In some embodiments, the thyroid hormone signaling pathwayactivator in the methods and composition provided herein comprises GC-1.In some embodiments, the thyroid hormone signaling pathway activator inthe methods and composition provided herein comprises an analog orderivative of T3 or GC-1. Exemplary analogs of T3 in the methods andcomposition provided herein include, but are not limited to, selectiveand non-selective thyromimetics, TRβ selective agonist-GC-1,GC-24,4-Hydroxy-PCB 106, MB07811, MB07344,3,5-diiodothyropropionic acid(DITPA); the selective TR-β agonist GC-1; 3-Iodothyronamine (T(1)AM) and3,3′,5-triiodothyroacetic acid (Triac) (bioactive metabolites of thehormone thyroxine (T(4)); KB-2115 and KB-141; thyronamines; SKF L-94901;DIBIT; 3′-AC-T2; tetraiodothyroacetic acid (Tetrac) andtriiodothyroacetic acid (Triac) (via oxidative deamination anddecarboxylation of thyroxine [T4] and triiodothyronine [T3] alaninechain), 3,3′,5′-triiodothyronine (rT3) (via T4 and T3 deiodination),3,3′-diiodothyronine (3,3′-T2) and 3,5-diiodothyronine (T2) (via T4, T3,and rT3 deiodination), and 3-iodothyronamine (T1AM) and thyronamine(T0AM) (via T4 and T3 deiodination and amino acid decarboxylation), aswell as for TH structural analogs, such as 3,5,3′-triiodothyropropionicacid (Triprop), 3,5-dibromo-3-pyridazinone-1-thyronine (L-940901),N-[3,5-dimethyl-4-(4′-hydroxy-3′-isopropylphenoxy)-phenyl]-oxamic acid(CGS 23425),3,5-dimethyl-4-[(4′-hydroxy-3′-isopropylbenzyl)-phenoxy]acetic acid(GC-1), 3,5-dichloro-4-[(4-hydroxy-3-isopropylphenoxy)phenyl]acetic acid(KB-141), and 3,5-diiodothyropropionic acid (DITPA).

In some embodiments, the thyroid hormone signaling pathway activator inthe methods and composition provided herein comprises a prodrug orprohormone of T3, such as T4 thyroid hormone (e.g., thyroxine orL-3,5,3′,5′-tetraiodothyronine).

In some embodiments, the thyroid hormone signaling pathway activator inthe methods and composition provided herein is an iodothyroninecomposition described in U.S. Pat. No. 7,163,918.

Epidermal Growth Factor (EGF) Family

Aspects of the disclosure relate to the use of growth factors from theEGF family as β cell differentiation factors.

In some embodiments, the at least one growth factor from the EGF familyin the methods and composition provided herein comprises betacellulin.In some embodiments, at least one growth factor from the EGF family inthe methods and composition provided herein comprises EGF. Epidermalgrowth factor (EGF) is a 53 amino acid cytokine which is proteolyticallycleaved from a large integral membrane protein precursor. In someembodiments, the growth factor from the EGF family in the methods andcomposition provided herein comprises a variant EGF polypeptide, forexample an isolated epidermal growth factor polypeptide having at least90% amino acid identity to the human wild-type EGF polypeptide sequence,as disclosed in U.S. Pat. No. 7,084,246. In some embodiments, the growthfactor from the EGF family in the methods and composition providedherein comprises an engineered EGF mutant that binds to and agonizes theEGF receptor, as is disclosed in U.S. Pat. No. 8,247,531. In someembodiments, the at least one growth factor from the EGF family in themethods and composition provided herein is replaced with an agent thatactivates a signaling pathway in the EGF family. In some embodiments,the growth factor from the EGF family in the methods and compositionprovided herein comprises a compound that mimics EGF. In certainembodiments, the methods, compositions, and kits disclosed hereinexclude a growth factor from the EGF family.

Protein Kinase Inhibitors

Aspects of the disclosure relate to the use of protein kinase inhibitorsas β cell differentiation factors.

In some embodiments, the protein kinase inhibitor in the methods andcomposition provided herein comprises staurosporine. In someembodiments, the protein kinase inhibitor in the methods and compositionprovided herein comprises an analog of staurosporine. Exemplary analogsof staurosporine in the methods and composition provided herein include,without limitation, Ro-31-8220, a bisindolylmaleimide (Bis) compound,10′-{5″-[(methoxycarbonyl)amino]-2″-methyl}-phenylaminocarbonylstaurosporine,a staralog (see, e.g., Lopez et al., “Staurosporine-derived inhibitorsbroaden the scope of analog-sensitive kinase technology”, J. Am. Chem.Soc. 2013; 135(48):18153-18159), and, cgp41251.

In some embodiments, the protein kinase inhibitor in the methods andcomposition provided herein is an inhibitor of PKCβ. In someembodiments, the protein kinase inhibitor in the methods and compositionprovided herein is an inhibitor of PKCβ with the following structure ora derivative, analogue or variant of the compound as follows:

In some embodiments, the inhibitor of PKCβ is a GSK-2 compound with thefollowing structure or a derivative, analogue or variant of the compoundas follows:

In some embodiments, the inhibitor of PKC in the methods and compositionprovided herein is a bisindolylmaleimide. Exemplary bisindolylmaleimidesinclude, without limitation, bisindolylmaleimide I, bisindolylmaleimideII, bisindolylmaleimide Ill, hydrochloride, or a derivative, analogue orvariant thereof.

In some embodiments, the PKC inhibitor in the methods and compositionprovided herein is a pseudohypericin, or a derivative, analogue, orvariant thereof. In some embodiments, the PKC inhibitor in the methodsand composition provided herein is indorublin-3-monoximc, 5-Iodo or aderivative, analogue or variant thereof. In certain embodiments, themethods, compositions, and kits disclosed herein exclude a proteinkinase inhibitor.

VII. SC-β Cells

The SC-β cells of the disclosure share many characteristic features of βcells which are important for normal β cell function. In someembodiments, the SC-β cell exhibits a glucose stimulated insulinsecretion (GSIS) response in vitro. In some embodiments, the SC-β cellexhibits a GSIS response in vivo. In some embodiments, the SC-β cellexhibits in vitro and in vivo GSIS responses. In some embodiments, theGSIS responses resemble the GSIS responses of an endogenous maturepancreatic β cell. In some embodiments, the SC-β cell exhibits a GSISresponse to at least one glucose challenge. In some embodiments, theSC-β cell exhibits a GSIS response to at least two sequential glucosechallenges. In some embodiments, the SC-β cell exhibits a GSIS responseto at least three sequential glucose challenges. In some embodiments,the GSIS responses resemble the GSIS response of endogenous human isletsto multiple glucose challenges. In some embodiments, the GSIS responseis observed immediately upon transplanting the cell into a human oranimal. In some embodiments, the GSIS response is observed withinapproximately 24 hours of transplanting the cell into a human or animal.In some embodiments, the GSIS response is observed within approximatelyone week of transplanting the cell into a human or animal. In someembodiments, the GSIS response is observed within approximately twoweeks of transplanting the cell into a human or animal. In someembodiments, the stimulation index of the cell as characterized by theratio of insulin secreted in response to high glucose concentrationscompared to low glucose concentrations is similar to the stimulationindex of an endogenous mature pancreatic β cell. In some embodiments,the SC-β cell exhibits a stimulation index of greater than 1. In someembodiments, the SC-β cell exhibits a stimulation index of greater thanor equal to 1. In some embodiments, the SC-β cell exhibits a stimulationindex of greater than 1.1. In some embodiments, the SC-β cell exhibits astimulation index of greater than or equal to 1.1. In some embodiments,the SC-β cell exhibits a stimulation index of greater than 2. In someembodiments, the SC-β cell exhibits a stimulation index of greater thanor equal to 1. In some embodiments, the SC-β cell exhibits a stimulationindex of at least 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, or 5.0 or greater.

Some embodiments of the present disclosure relate to cell compositions,such as cell cultures or cell populations, comprising SC-β cells,wherein the SC-β cells have been derived from at least oneinsulin-positive endocrine cell or a precursor thereof. In someembodiments, the cell compositions comprise insulin-positive endocrinecells. In some embodiments, the cell compositions compriseNKX6.1-pancreatic progenitor cells. In some embodiments, the cellcompositions comprise PDX1-pancreatic progenitor cells. In someembodiments, the cell compositions comprise primitive gut tube cells. Insome embodiments, the cell compositions comprise definitive endodermcells.

In accordance with certain embodiments, the chemically induced SC-βcells are mammalian cells, and in a preferred embodiment, such SC-βcells are human SC-β cells. In some embodiments, the insulin-positiveendocrine cells have been derived from definitive endoderm cells e.g.human definitive endoderm stem cells. In accordance with certainembodiments, the chemically induced PDX1-positive pancreatic progenitorsare mammalian cells, and in a preferred embodiment, such PDX1-positivepancreatic progenitors are human PDX1-positive pancreatic progenitors.

Other embodiments of the present disclosure relate to compositions, suchas an isolated cell population or cell culture, comprising SC-β cellsproduced by the methods as disclosed herein. In some embodiments of thepresent disclosure relate to compositions, such as isolated cellpopulations or cell cultures, comprising chemically-induced SC-β cellsproduced by the methods as disclosed herein. In such embodiments, theSC-β cells comprise less than about 90%, less than about 85%, less thanabout 80%, less than about 75%, less than about 70%, less than about65%, less than about 60%, less than about 55%, less than about 50%, lessthan about 45%, less than about 40%, less than about 35%, less thanabout 30%, less than about 25%, less than about 20%, less than about15%, less than about 12%, less than about 10%, less than about 8%, lessthan about 6%, less than about 5%, less than about 4%, less than about3%, less than about 2% or less than about 1% of the total cells in theSC-β cells population. In some embodiments, the composition comprises apopulation of SC-β cells which make up more than about 90% of the totalcells in the cell population, for example about at least 95%, or atleast 96%, or at least 97%, or at least 98% or at least about 99%, orabout at least 100% of the total cells in the cell population are SC-βcells.

Certain other embodiments of the present disclosure relate tocompositions, such as an isolated cell population or cell cultures,comprise a combination of SC-β cells and insulin-positive endocrinecells or precursors thereof from which the SC-β cells were derived. Insome embodiments, the insulin-positive endocrine cells from which theSC-β cells are derived comprise less than about 25%, less than about20%, less than about 15%, less than about 10%, less than about 5%, lessthan about 4%, less than about 3%, less than about 2% or less than about1% of the total cells in the isolated cell population or culture.

Additional embodiments of the present disclosure relate to compositions,such as isolated cell populations or cell cultures, produced by theprocesses described herein and which comprise chemically induced SC-βcells as the majority cell type. In some embodiments, the methods andprocesses described herein produces an isolated cell culture and/or cellpopulations comprising at least about 99%, at least about 98%, at leastabout 97%, at least about 96%, at least about 95%, at least about 94%,at least about 93%, at least about 92%, at least about 91%, at leastabout 90%, at least about 89%, at least about 88%, at least about 87%,at least about 86%, at least about 85%, at least about 84%, at leastabout 83%, at least about 82%, at least about 81%, at least about 80%,at least about 79%, at least about 78%, at least about 77%, at leastabout 76%, at least about 75%, at least about 74%, at least about 73%,at least about 72%, at least about 71%, at least about 70%, at leastabout 69%, at least about 68%, at least about 67%, at least about 66%,at least about 65%, at least about 64%, at least about 63%, at leastabout 62%, at least about 61%, at least about 60%, at least about 59%,at least about 58%, at least about 57%, at least about 56%, at leastabout 55%, at least about 54%, at least about 53%, at least about 52%,at least about 51% or at least about 50% SC-β cells.

In another embodiment, isolated cell populations or compositions ofcells (or cell cultures) comprise human SC-β cells. In otherembodiments, the methods and processes as described herein can produceisolated cell populations comprising at least about 50%, at least about45%, at least about 40%, at least about 35%, at least about 30%, atleast about 25%, at least about 24%, at least about 23%, at least about22%, at least about 21%, at least about 20%, at least about 19%, atleast about 18%, at least about 17%, at least about 16%, at least about15%, at least about 14%, at least about 13%, at least about 12%, atleast about 11%, at least about 10%, at least about 9%, at least about8%, at least about 7%, at least about 6%, at least about 5%, at leastabout 4%, at least about 3%, at least about 2% or at least about 1% SC-βcells. In preferred embodiments, isolated cell populations can comprisehuman SC-β cells. In some embodiments, the percentage of SC-β cells inthe cell cultures or populations is calculated without regard to thefeeder cells remaining in the culture.

Yet another aspect of the present disclosure relates to cell populationsor compositions of cells (or cell cultures) that comprise at least about50%, at least about 45%, at least about 40%, at least about 35%, atleast about 30%, at least about 25%, at least about 24%, at least about23%, at least about 22%, at least about 21%, at least about 20%, atleast about 19%, at least about 18%, at least about 17%, at least about16%, at least about 15%, at least about 14%, at least about 13%, atleast about 12%, at least about 11%, at least about 10%, at least about9%, at least about 8%, at least about 7%, at least about 6%, at leastabout 5%, at least about 4%, at least about 3%, at least about 2% or atleast about 1% NKX6.1⁺/C-peptide⁺ cells. In some embodiments, the cellpopulation or composition of cells as provided herein comprises at leastabout 20% NKX6.1⁺/C-peptide⁺ cells. In some embodiments, the cellpopulation or composition of cells as provided herein comprises at leastabout 40% NKX6.1⁺/C-peptide⁺ cells. In some embodiments, the cellpopulation or composition of cells as provided herein comprises at leastabout 50% NKX6.1⁺/C-peptide⁺ cells. In some embodiments, the cellpopulation or composition of cells as provided herein comprises about17.9% NKX6.1⁺/C-peptide⁺ cells. In some embodiments, the cell populationor composition of cells as provided herein comprises about 41.5%NKX6.1⁺/C-peptide⁺ cells. In some embodiments, the cell population orcomposition of cells as provided herein comprises about 50.6%NKX6.1⁺/C-peptide⁺ cells.

In some embodiments, the cell population or composition of cells asprovided herein comprises at least about 90%, at least about 89%, atleast about 88%, at least about 85%, at least about 80%, at least about75%, at least about 70%, at least about 65%, at least about 60%, atleast about 55%, at least about 50%, at least about 45%, at least about40%, at least about 35%, at least about 30%, at least about 25%, atleast about 24%, at least about 23%, at least about 22%, at least about21%, at least about 20%, at least about 19%, at least about 18%, atleast about 17%, at least about 16%, at least about 15%, at least about14%, at least about 13%, at least about 12%, at least about 11%, atleast about 10%, at least about 9%, at least about 8%, at least about7%, at least about 6%, at least about 5%, at least about 4%, at leastabout 3%, at least about 2% or at least about 1% NKX6.1⁺ cells. In someembodiments, the cell population or composition of cells as providedherein comprises at least about 40% NKX6.1⁺ cells. In some embodiments,the cell population or composition of cells as provided herein comprisesat least about 60% NKX6.1⁺ cells. In some embodiments, the cellpopulation or composition of cells as provided herein comprises at leastabout 85% NKX6.1⁺ cells. In some embodiments, the cell population orcomposition of cells as provided herein comprises about 36.9% NKX6.1⁺cells. In some embodiments, the cell population or composition of cellsas provided herein comprises about 63.4% NKX6.1⁺ cells. In someembodiments, the cell population or composition of cells as providedherein comprises about 89.5% NKX6.1⁺ cells.

In some embodiments, the cell population or composition of cells asprovided herein comprises at least about 55%, at least about 50%, atleast about 45%, at least about 40%, at least about 35%, at least about30%, at least about 25%, at least about 24%, at least about 23%, atleast about 22%, at least about 21%, at least about 20%, at least about19%, at least about 18%, at least about 17%, at least about 16%, atleast about 15%, at least about 14%, at least about 13%, at least about12%, at least about 11%, at least about 10%, at least about 9%, at leastabout 8%, at least about 7%, at least about 6%, at least about 5%, atleast about 4%, at least about 3%, at least about 2% or at least about1% C-peptide⁺ cells. In some embodiments, the cell population orcomposition of cells as provided herein comprises at least about 30%C-peptide⁺ cells. In some embodiments, the cell population orcomposition of cells as provided herein comprises at least about 55%C-peptide⁺ cells. In some embodiments, the cell population orcomposition of cells as provided herein comprises about 26.8% C-peptide⁺cells. In some embodiments, the cell population or composition of cellsas provided herein comprises about 57.7% C-peptide⁺ cells. In someembodiments, the cell population or composition of cells as providedherein comprises about 55.2% C-peptide⁺ cells.

In some embodiments, the cell population or composition of cells asprovided herein comprises at least about 99%, at least about 98%, atleast about 95%, at least about 90%, at least about 89%, at least about88%, at least about 85%, at least about 80%, at least about 75%, atleast about 70%, at least about 65%, at least about 60%, at least about55%, at least about 50%, at least about 45%, at least about 40%, atleast about 35%, at least about 30%, at least about 25%, at least about24%, at least about 23%, at least about 22%, at least about 21%, atleast about 20%, at least about 19%, at least about 18%, at least about17%, at least about 16%, at least about 15%, at least about 14%, atleast about 13%, at least about 12%, at least about 11%, at least about10%, at least about 9%, at least about 8%, at least about 7%, at leastabout 6%, at least about 5%, at least about 4%, at least about 3%, atleast about 2% or at least about 1% Chromogranin A (CHGA)⁺ cells. Insome embodiments, the cell population or composition of cells asprovided herein comprises at least about 40% CHGA⁺ cells. In someembodiments, the cell population or composition of cells as providedherein comprises at least about 85% CHGA⁺ cells. In some embodiments,the cell population or composition of cells as provided herein comprisesat least about 95% CHGA⁺ cells. In some embodiments, the cell populationor composition of cells as provided herein comprises about 37.7% CHGA⁺cells. In some embodiments, the cell population or composition of cellsas provided herein comprises about 87.5% CHGA⁺ cells. In someembodiments, the cell population or composition of cells as providedherein comprises about 96.4% CHGA⁺ cells.

Still other embodiments of the present disclosure relate tocompositions, such as isolated cell populations or cell cultures,comprising mixtures of SC-β cells and insulin-positive endocrine cellsor precursors thereof from which they were differentiated from. Forexample, cell cultures or cell populations comprising at least about 5SC-β cells for about every 95 insulin-positive endocrine cells orprecursors thereof can be produced. In other embodiments, cell culturesor cell populations comprising at least about 95 SC-β cells for aboutevery 5 insulin-positive endocrine cells or precursors thereof can beproduced. Additionally, cell cultures or cell populations comprisingother ratios of SC-β cells to insulin-positive endocrine cells orprecursors thereof are contemplated. For example, compositionscomprising at least about 1 SC-β cell for about every 1,000,000, or atleast 100,000 cells, or at least 10,000 cells, or at least 1000 cells or500, or at least 250 or at least 100 or at least 10 insulin-positiveendocrine cells or precursors thereof can be produced.

In some aspects, the present disclosure provides a cell clustercomprising at least about 50% Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells, at most about 30%, 28%, 26%, 25%, 24%, 22%, 20%, 18%,16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% chromogranin A(CHGA)-positive cells, and at most about 30%, 28%, 26%, 25%, 24%, 22%,20%, 18%, 16%, or 15% CDX2-positive cells. In some cases, the cellcluster comprises at most about 20% the CDX2-positive, NKX6.1-positivecells. In some cases, the cell cluster comprises at most about 5% theCHGA-positive cells. In some embodiments, the cell cluster comprises atmost about 20% the CDX2-positive, NKX6.1-positive cells and at mostabout 5% the CHGA-positive cells. In some embodiments, the cell clustercomprises at least about 60%, 62%, 64%, 65%, 68%, 70%, 72%, 74%, 76%,78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, or 95% the Pdx1-positive,NKX6.1-positive pancreatic progenitor cells. In some embodiments, thecell cluster comprises at least about 65% the Pdx1-positive,NKX6.1-positive pancreatic progenitor cells.

In some embodiments, the cell cluster comprising at least about 50%Pdx1-positive, NKX6.1-positive pancreatic progenitor cells, at mostabout 30% chromogranin A (CHGA)-positive cells, and at most about 30%CDX2-positive cells can have particular functional features as comparedto a comparable cell cluster having more than about 30% chromogranin A(CHGA)-positive cells or more than about 30% CDX2-positive cells. Forinstance, in some cases, further differentiation of the cell clustercomprising at least about 50% Pdx1-positive, NKX6.1-positive pancreaticprogenitor cells, at most about 30% chromogranin A (CHGA)-positivecells, and at most about 30% CDX2-positive cells results in a first cellcluster comprising non-native pancreatic β cells that has a higherglucose-stimulated insulin secretion (GSIS) stimulation index than asecond cell cluster comprising the non-native pancreatic β cellsdifferentiated from a comparable cell cluster comprising at least about50% the Pdx1-positive, NKX6.1-positive pancreatic progenitor cells, andmore than 30% the chromogranin A (CHGA)-positive cells or more than 30%the CDX2-positive cells as measured by flow cytometry.

In some aspects, the present disclosure provides a cell clustercomprising at least about 60%, 62%, 64%, 65%, 68%, 70%, 72%, 74%, 76%,78%, 80%, 82%, 84%, 86%, 88%, or 90% Pdx1-positive, NKX6.1-negativepancreatic progenitor cells and at most about 40%, 38%, 36%, 34%, 32%,30%, 28%, 26%, 25%, 24%, 22%, 20%, 18%, 16%, 15%, 14%, 12%, or 10%CDX2-positive cells. In some embodiments, the cell cluster comprises atleast about 85% the Pdx1-positive, NKX6.1-negative pancreatic progenitorcells. In some embodiments, the cell cluster comprises at most about 15%the CDX2-positive cells. In some cases, the cell cluster comprises atleast about 85% the Pdx1-positive, NKX6.1-negative pancreatic progenitorcells and at most about 15% the CDX2-positive cells.

In some embodiments, the cell cluster comprising at least about 60%Pdx1-positive, NKX6.1-negative pancreatic progenitor cells and at mostabout 40% CDX2-positive cells can have particular functional features ascompared to a comparable cell cluster having more than about 40%CDX2-positive cells. For instance, in some cases, furtherdifferentiation of the cell cluster comprising at least about 60%Pdx1-positive, NKX6.1-negative pancreatic progenitor cells and at mostabout 40% CDX2-positive cells results in a first cell cluster comprisingnon-native pancreatic β cells that has a higher glucose-stimulatedinsulin secretion (GSIS) stimulation index than a second cell clustercomprising the non-native pancreatic β cells differentiated from acomparable cell cluster comprising at least about 60% Pdx1-positive,NKX6.1-negative pancreatic progenitor cells and more than 40% theCDX2-positive cells as measured by flow cytometry.

In some aspects, the present disclosure provides a cell clustercomprising non-native pancreatic β cells. In some embodiments, the cellcluster disclosed herein is obtained from differentiation of primitivegut tube cells by contacting the primitive gut tube cells with a bonemorphogenetic protein (BMP) signaling pathway inhibitor and a growthfactor from transformation growth factor β (TGF-β) superfamily. In someembodiments, the cell cluster has a higher number of the non-nativepancreatic β cells per cubic micrometer as compared to a comparablesecond cell cluster obtained from differentiation of primitive gut tubecells without the contacting. In some embodiments, cell cluster has anat least about 1.1, 1.2, 1.3, 1.4, 1.5, or 1.6 fold higher number of thenon-native pancreatic β cells per cubic micrometer as compared to thecomparable second cell cluster.

In some cases, the cell cluster comprising non-native pancreatic β cellsdisclosed herein exhibits higher insulin secretion in response toglucose challenge as compared to a comparable cell cluster obtained fromdifferentiation of primitive gut tube cells without contacting with BMPsignaling pathway inhibitor or growth factor from TGF-β family. In someembodiments, the cell cluster exhibits at least about 1.2, 1.5, 2, 2.5,3, 3.5, 4, 4.5, or 5 fold higher an insulin secretion as compared to thecomparable second cell cluster. In some embodiments, the cell clusterexhibits a higher GSIS stimulation index as compared to the comparablesecond cell cluster. In some embodiments, the GSIS stimulation index ofthe cell cluster is at least about 1.2 fold, at least about 1.5 fold, atleast about 1.8 fold, at least about 2 fold, at least about 2.2 fold, atleast about 2.4 fold, at least about 2.8 fold, or at least about 3 foldhigher than that of the second cell cluster. In some embodiments, GSISstimulation index of the cell cluster is at least about 3 fold higherthan that of the second population. In some embodiments, GSISstimulation index is calculated as a ratio of insulin secretion inresponse to 20 mM glucose challenge to insulin secretion in response to2 mM glucose challenge. In some embodiments, the non-native pancreatic βcells exhibit an in vitro glucose-stimulated insulin secretion responsewhen exposed to a glucose challenge. In some cases, non-nativepancreatic β cells exhibit an insulin secretion in response to a firstconcentration of K⁺. In some embodiments, the cell cluster exhibits ahigher insulin secretion as compared to the comparable second cellcluster in response to a first concentration of K⁺. In some embodiments,cell cluster exhibits at least about 1.2 fold, at least about 1.5 fold,at least about 1.8 fold, at least about 2 fold, at least about 2.2 fold,at least about 2.4 fold, at least about 2.8 fold, at least about 3 fold,at least about 3.2 fold, at least about 3.4 fold, at least about 3.6fold, at least about 3.8 fold, at least about 4 fold higher an insulinsecretion as compared to the comparable second cell cluster in responseto a first concentration of K⁺.

In some cases, cell populations or cell clusters disclosed herein areunsorted, e.g., isolated cell populations or cell clusters that have notbeen through cell sorting process. In some embodiments, the cell clusterdisclosed herein can refer to a cell cluster formed by self-aggregationof cells cultured in a given environment, for instance, in a 3Dsuspension culture. In some embodiments, cell clusters disclosed hereinare intermediate cell clusters formed during the differentiation processas described herein. In some cases, the intermediate cell clusters,e.g., cell clusters comprising Pdx1-positive, NKX6.1-negative pancreaticprogenitor cells (e.g., Stage 3 cell clusters) or cell clusterscomprising Pdx1-positive, NKX6.1-positive pancreatic progenitor cells(e.g., Stage 4 cell clusters), are not subjected to cell sorting. Insome case, cell populations going through cell sorting may not be ableto form the intermediate cell clusters disclosed herein. For instance,Pdx1-positive pancreatic progenitor cells, after going through cellsorting, may not be able to form a cell cluster as disclosed herein.

Cell sorting as described herein can refer to a process of isolating agroup of cells from a plurality of cells by relying on differences incell size, shape (morphology), surface protein expression, endogenoussignal protein expression, or any combination thereof. In some cases,cell sorting comprises subjecting the cells to flow cytometry. Flowcytometry can be a laser- or impedance-based, biophysical technology.During flow cytometry, one can suspend cells in a stream of fluid andpass them through an electronic detection apparatus. In one type of flowcytometry, fluorescent-activated cell sorting (FACS), based on one ormore parameters of the cells' optical properties (e.g., emission wavelength upon laser excitation), one can physically separate and therebypurify cells of interest using flow cytometry. As described herein, anunsorted cell cluster can be cell cluster that formed by a plurality ofcells that have not been subject to an active cell sorting process,e.g., flow cytometry. In some cases, flow cytometry as discussed hereincan be based on one or more signal peptides expressed in the cells. Forexample, a cell cluster can comprise cells that express a signal peptide(e.g., a fluorescent protein, e.g., green fluorescent protein (GFP) ortdTomato). In some cases, the signal peptide is expressed as anindicator of insulin expression in the cells. For instance, a cellcluster can comprise cell harboring an exogenous nucleic acid sequencecoding for GFP under the control of an insulin promoter. The insulinpromoter can be an endogenous or exogenous promoter. In some cases, theexpression of GFP in these cells can be indicative of insulin expressionin the cells. The GFP signal can thus be a marker of a pancreatic βcell. In some cases, cell sorting as described herein can comprisemagnetic-activated flow cytometry, where magnetic antibody or otherligand is used to label cells of different types, and the differences inmagnetic properties can be used for cell sorting.

The percentage of cells expressing one or more particular markers, likePdx1, NKX6.1, insulin, NGN3, or CHGA, described herein can be thepercentage value detected using techniques like flow cytometry assay. Insome cases, during a flow cytometry assay, cell population or cellcluster discussed herein are dispersed into single-cell suspension byincubation in digesting enzyme like trypsin or TrypLE™ Express.Dispersed cell can be washed in suitable buffer like PBS, centrifugedand then re-suspended in fixation buffer like 4% PFA. Incubation withprimary antibodies against the cell markers of interest can then beconducted, which can be followed by incubation with the secondaryantibodies. After antibody incubation, the cells can be washed and thesubject to segregation by flow cytometry. Techniques other than flowcytometry can also be used to characterize the cells described herein,e.g., determine the cell percentages. Non-limiting examples of cellcharacterization methods include gene sequencing, microscopic techniques(fluorescence microscopy, atomic force microscopy), karyotyping,isoenzyme analysis, DNA properties, viral susceptibility.

In some aspects, the disclosure relates to a composition comprising apopulation of glucose-responsive insulin secreting cells, wherein thecells secrete a higher amount of insulin upon induction with KCl (e.g.,about 20 to about 50 mM, e.g., about 30 mM) as compared to the amount ofinsulin secreted upon induction with glucose. In some embodiments, thepopulation of glucose-responsive insulin secreting cells secrete atleast 1.5 times, 2 times, 2.5 times, 3 times higher amount of insulinupon induction with KCl as compared to the amount of insulin secretedupon induction with glucose.

In some aspects, the disclosure relates to a composition comprising apopulation of glucose-responsive insulin secreting cells, wherein thecells secrete a higher amount of insulin upon induction with KCl and/orglucose, in the presence of a signaling factor as compared to comparablecells in the absence of the signaling factor. In some embodiments, thecells secrete higher amount of insulin in the presence of high glucose,but not in the presence of low glucose. In some embodiments, the highglucose concentration is about 10-20 mM. In some embodiments, the lowglucose concentration is about 2-5 mM.

In some aspects, the disclosure relates to a composition comprising apopulation of differentiated pancreatic progenitor cells, wherein thepopulation comprises at least 60% pancreatic β cells as determined byflow cytometry. In some embodiments, the population comprises at least65%, 70%, 75%, 80%, 85%, or 90% pancreatic β cells. In some embodiments,the population comprises a higher percentage of pancreatic β cells uponbeing contacted with a predetermined basal medium component as comparedto a comparable population not contacted with the basal mediumcomponent.

The in vitro-matured, SC-β cell (e.g., pancreatic β cells) generatedaccording to the disclosed methods described herein demonstrate manyadvantages, for example, they perform glucose stimulated insulinsecretion in vitro, resemble human islet β cells by gene expression andultrastructure, secrete human insulin and ameliorate hyperglycemia whentransplanted into mice, provide a new platform for cell therapy (e.g.,transplantation into a subject in need of additional and/or functional βcells), drug screening (e.g., for insulin production/secretion,survival, dedifferentiation, etc.), research (e.g., determining thedifferences in function between normal and diabetic β cell), and tissueengineering (e.g., using the SC-β cells as the first cell type inreconstructing an islet).

VIII. Methods of Reducing Proliferation

Provided herein is a method to reduce proliferation in cell populationof SC-β cells that is generated according to the methods describedherein. A method can comprise irradiating an in vitro cell populationcomprising endocrine cells. In some cases, the irradiation of the cellpopulation comprising endocrine cells is at a dose of about 100 rads toabout 100,000 rads for a time period of about 1 min to about 60 min.

In some cases, the irradiation of the cell population comprisingendocrine cells is at a dose of about 100 rads to about 50,000 rads,about 100 rads to about 25,000 rads, about 100 rads to about 10,000rads, about 250 rads to about 25,000 rads, about 500 rads to about25,000 rads, about 1,000 rads to about 25,000 rads, about 2,500 rads toabout 25,000 rads, about 5,000 rads to about 25,000 rads, or about10,000 rads to about 15,000 rads. In some cases, the irradiation of thecell population comprising endocrine cells is at a dose of about 10,000rads. In some cases, the irradiation of the cell population comprisingendocrine cells is conducted for about 1 min to about 55 min, about 1min to about 50 min, about 1 min to about 45 min, about 1 min to about40 min, about 1 min to about 35 min, about 1 min to about 30 min, about1 min to about 25 min, about 1 min to about 20 min, about 1 min to about10 min, about 1 min to about 5 min, about 10 min to about 55 min, about15 min to about 55 min, about 20 min to about 55 min, about 25 min toabout 55 min, about 30 min to about 55 min, about 20 min to about 40min, or about 25 min to about 35 min. In some cases, the irradiation ofthe cell population comprising endocrine cells is for about 30 min.

As used herein, irradiation can refer to ionizing irradiation. It can beconducted by exposing the cell population to gamma rays, x-rays,ultraviolet radiation, alpha rays, beta rays (electron beams), orneutron rays. Without wishing to be bound by a certain theory, theionizing irradiation can control cell growth by damaging DNA of cells,e.g., proliferating cells, and consequentially cell death. In somecases, the cell population comprising endocrine cells that has beensubject to the irradiation as described herein has a lower proportion ofcells capable of proliferation or proliferating cells as compared to acorresponding cell population that has not been subject to theirradiation. In some cases, there is at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%,300%, 320%, 350%, 380%, 400%, 420%, 450%, 480%, or 500% lower proportionof cells capable of proliferation or proliferating cells in the cellpopulation comprising endocrine cells that has been exposed to theirradiation as compared to a corresponding cell population that has notbeen exposed to the irradiation.

In some cases, the methods provided herein can comprise exposing toirradiation a cell population during pancreatic differentiation thatcomprises stem cells, definitive endoderm cells, primitive gut tubecells, pancreatic progenitor cells, or endocrine cells. In some cases,the irradiation results in a cell population that has reducedproliferative capability as compared to a corresponding cell populationthat is not subject to irradiation.

In some cases, the cell population comprising endocrine cells is a cellpopulation obtained via any of the differentiation methods providedherein. In some cases, the cell population comprising endocrine cells isa cell population obtained by the stepwise differentiation methodsprovided herein. In some cases, the cell population comprising endocrinecells is a cell population at Stage 6 of the differentiation protocol.In some cases, the cell population comprising endocrine cells is a cellpopulation on day 1 at Stage 6 (S6d1), S6d2, S6d3, S6d4, S6d5, S6d6, orS6d7. In some cases, the cell population comprising endocrine cells is acell population thawed and recovered from cryopreservation. In somecases, the cell population comprising endocrine cells is cryopreservedwhile being exposed to the irradiation. The cell population comprisingendocrine cells can be thawed and recovered for further differentiationinto pancreatic β cells after exposure to the irradiation as describedherein.

In some cases, the cell population comprising endocrine cells can befurther differentiated/matured into a cell population comprisingpancreatic β cells, which can have a lower proportion of cells capableof proliferation or proliferating cells as compared to a correspondingcell population without being the same exposure to the irradiation. Insome cases, there is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 120%, 150%, 180%, 200%, 220%, 250%, 280%, 300%, 320%,350%, 380%, 400%, 420%, 450%, 480%, or 500% lower proportion of cellscapable of proliferation or proliferating cells in the cell populationcomprising pancreatic β cells that is obtained from the irradiated cellpopulation comprising endocrine as compared to a corresponding cellpopulation that has not been exposed to the irradiation.

In some cases, the cell population comprising pancreatic β cells that isobtained from the irradiated cell population comprising endocrine isimplanted into as subject in need thereof. The cell populationcomprising pancreatic β cells can help control the blood glucose levelin the subject. In some cases, the cell population comprising pancreaticβ cells that is obtained from the irradiated cell population comprisingendocrine can maintain the blood glucose control in the subject for atleast about 50 days, 60 days, 70 days, 80 days, 90 days, or even longer.In some cases, the cell population comprising pancreatic β cells that isobtained from the irradiated cell population comprising endocrine canmaintain the blood glucose control in the subject for 60 days. In somecases, the cell population comprising pancreatic β cells that isobtained from the irradiated cell population comprising endocrine canmaintain the blood glucose control in the subject for 90 days.

IX. Methods of Enriching Stem Cell Derived Beta Cells

Provided herein is a method to isolate of a population of SC-β cellsfrom a heterogeneous population of cells, such a mixed population ofcells comprising SC-β cells and insulin-positive endocrine cells orprecursors thereof from which the SC-β cells were derived. A populationof SC-β cells produced by any of the above-described processes can beenriched, isolated and/or purified by using any cell surface marker,e.g., polysialylated-neural cell adhesion molecule (PSA-NCAM), presenton the SC-β cells which is not present on the insulin-positive endocrinecell or precursor thereof from which it was derived. Such cell surfacemarkers are also referred to as an affinity tag which is specific for aSC-β cell.

In some embodiments, the cell surface marker is an inducible cellsurface marker. For example, PSA-NCAM can be induced to the surface bycertain signals. Different types of endocrine cells can respond todifferent signals. In some embodiments, PSA-NCAM can be selectivelycleaved off the surface by an enzyme, e.g., endoneuraminidase or Endo-N.Endo-N is an endosialidase which degrades rapidly and specificallylinear polymers of sialic acid with α-2,8-linkage with a minimum lengthof 7-9 residues characteristic of sialic acid residues associated withNCAM.

Cell Sorting

The methods provided herein relates to a cell composition comprising afirst population of endocrine cells and a second population of endocrinecells, wherein a reduced proportion of cells of the first population ofendocrine cells express VMAT⁺ or Cdx2⁺ as compared to a secondpopulation of endocrine cells. In some embodiments, the first and secondpopulations of endocrine cells comprise beta cells which can be inducedby glucose and stem cells (e.g., hPSC or EC). In some embodiments, thestem cells are induced by isoproterenol. In some embodiments, thepopulations of cells comprise a mixture of cells that are inducedinsulin producing beta cells and non-insulin producing cells. In someembodiments, PSA-NCAM can be cleaved off from all cells to create a“blank slate”, can be induced to the cell surface with a cell typespecific stimulant resulting in only one cell type having PSA-NCAM onthe surface in large quantities, and can be used to selectively sortusing an affinity tag, e.g., an anti-PSA-NCAM antibody, for the inducedSC-β cells. In this manner, the differentiated SC-β cells can be sortedand enriched from other endocrine cells in the population of endocrinecells. SC-β cells. In some embodiments, one or more cells of thepopulation of cells fails to localize the selectable marker to a cellsurface when contacted with the stimulating compound.

Examples of affinity tags specific for SC-β cells are antibodies (e.g.,an anti-PSA-NCAM antibody), ligands or other binding agents that arespecific to a marker molecule, such as a polypeptide, that is present onthe cell surface of a SC-β cells but which is not substantially presenton other cell types (e.g. insulin-positive endocrine cells or precursorsthereof). In some processes, an antibody which binds to a cell surfaceantigen on a SC-β cell (e.g. a human SC-β cell) is used as an affinitytag for the enrichment, isolation or purification of chemically induced(e.g. by contacting with at least one β cell maturation factor asdescribed herein) SC-β cells produced by the methods described herein.Such antibodies are known and commercially available.

The skilled artisan will readily appreciate the processes for usingantibodies for the enrichment, isolation and/or purification of SC-βcell. For example, in some embodiments, the reagent, such as anantibody, is incubated with a cell population comprising SC-β cells,wherein the cell population has been treated to reduce intercellular andsubstrate adhesion. The cell population are then washed, centrifuged andresuspended. In some embodiments, if the antibody is not already labeledwith a label, the cell suspension is then incubated with a secondaryantibody, such as an FACS-conjugated antibody that is capable of bindingto the primary antibody. The SC-β cells are then washed, centrifuged andresuspended in buffer. The SC-β cell suspension is then analyzed andsorted using a fluorescence activated cell sorter (FACS).Antibody-bound, fluorescent reprogrammed cells are collected separatelyfrom non-bound, non-fluorescent cells (e.g. immature, insulin-producingcells), thereby resulting in the isolation of SC-β cells from othercells present in the cell suspension, e.g. insulin-positive endocrinecells or precursors thereof, or immature, insulin-producing cell (e.g.other differentiated cell types).

In another embodiment of the processes described herein, the isolatedcell composition comprising SC-β cells can be further purified by usingan alternate affinity-based method or by additional rounds of sortingusing the same or different markers that are specific for SC-β cells.For example, in some embodiments, FACS sorting is used to first isolatea SC-β cell which expresses NKX6-1, either alone or with the expressionof C-peptide, or alternatively with a β cell marker disclosed hereinfrom cells that do not express one of those markers (e.g. negativecells) in the cell population. A second FAC sorting, e.g. sorting thepositive cells again using FACS to isolate cells that are positive for adifferent marker than the first sort enriches the cell population forreprogrammed cells. In an alternative embodiment, FACS sorting is usedto separate cells by negatively sorting for a marker that is present onmost insulin-positive endocrine cells or precursors thereof but is notpresent on SC-β cells.

In some embodiments of the processes described herein, SC-β cells arefluorescently labeled without the use of an antibody then isolated fromnon-labeled cells by using a fluorescence activated cell sorter (FACS).In such embodiments, a nucleic acid encoding GFP, YFP or another nucleicacid encoding an expressible fluorescent marker gene, such as the geneencoding luciferase, is used to label reprogrammed cells using themethods described above. For example, in some embodiments, at least onecopy of a nucleic acid encoding GFP or a biologically active fragmentthereof is introduced into at least one insulin-positive endocrine cellwhich is first chemically induced into a SC-β cell, where a downstreamof a promoter expressed in SC-β cell, such as the insulin promoter, suchthat the expression of the GFP gene product or biologically activefragment thereof is under control of the insulin promoter.

In addition to the procedures just described, chemically induced SC-βcells may also be isolated by other techniques for cell isolation.Additionally, SC-β cells may also be enriched or isolated by methods ofserial subculture in growth conditions which promote the selectivesurvival or selective expansion of the SC-β cell. Such methods are knownby persons of ordinary skill in the art, and may include the use ofagents such as, for example, insulin, members of the TGF-beta family,including Activin A, TGF-beta1, 2, and 3, bone morphogenic proteins(BMP-2, -3, -4, -5, -6, -7, -11, -12, and -13), fibroblast growthfactors-1 and -2, platelet-derived growth factor-AA, and —BB, plateletrich plasma, insulin-like growth factors (IGF-I, II) growthdifferentiation factor (GDF-5, -6, -7, -8, -10, -11, -15), vascularendothelial cell-derived growth factor (VEGF), Hepatocyte growth factor(HGF), pleiotrophin, endothelin, Epidermal growth factor (EGF),beta-cellulin, among others. Other pharmaceutical compounds can include,for example, nicotinamide, glucagon like peptide-1 (GLP-1) and II, GLP-1and 2 mimetibody, Exendin-4, retinoic acid, parathyroid hormone.

Using the methods described herein, enriched, isolated and/or purifiedpopulations of SC-β cells can be produced in vitro from insulin-positiveendocrine cells or precursors thereof (which were differentiated frompluripotent stem cells by the methods described herein). In someembodiments, preferred enrichment, isolation and/or purification methodsrelate to the in vitro production of human SC-β cell from humaninsulin-positive endocrine cells or precursors thereof, which weredifferentiated from human pluripotent stem cells, or from human inducedpluripotent stem (iPS) cells. In such an embodiment, where SC-β cellsare differentiated from insulin-positive endocrine cells, which werepreviously derived from definitive endoderm cells, which were previouslyderived from iPS cells, the SC-β cell can be autologous to the subjectfrom whom the cells were obtained to generate the iPS cells.

Using the methods described herein, isolated cell populations of SC-βcells are enriched in SC-β cell content by at least about 2- to about1000-fold as compared to a population of cells before the chemicalinduction of the insulin-positive endocrine cell or precursorpopulation. In some embodiments, SC-β cells can be enriched by at leastabout 5- to about 500-fold as compared to a population before thechemical induction of an insulin-positive endocrine cell or precursorpopulation. In other embodiments, SC-β cells can be enriched from atleast about 10- to about 200-fold as compared to a population before thechemical induction of insulin-positive endocrine cell or precursorpopulation. In still other embodiments, SC-β cell can be enriched fromat least about 20- to about 100-fold as compared to a population beforethe chemical induction of insulin-positive endocrine cell or precursorpopulation. In yet other embodiments, SC-β cell can be enriched from atleast about 40- to about 80-fold as compared to a population before thechemical induction of insulin-positive endocrine cell or precursorpopulation. In certain embodiments, SC-β cell can be enriched from atleast about 2- to about 20-fold as compared to a population before thechemical induction of insulin-positive endocrine cell or precursorpopulation.

Provided herein is a method of selecting a target cell (e.g., SC-β cell)from a population of cells comprising contacting the target cell with astimulating compound, wherein the contacting induces a selectable marker(e.g., PSA-NCAM) of the target cell to localize to a cell surface of thetarget cell, and selecting the target cell (e.g., SC-β cell) based onthe localization of the selectable marker (e.g., PSA-NCAM) at the cellsurface. In some embodiments, the selectable marker comprises PSA-NCAM.In some embodiments, the selecting the target cell is by cell sorting.In some embodiments, the selecting comprises contacting the selectablemarker of the target cell with an antigen binding polypeptide when theselectable marker is localized to the surface of the target cell. Insome embodiments, the antigen binding polypeptide comprises an antibody.In some embodiments, the antigen binding polypeptide binds to thePSA-NCAM. In some embodiments, the method further comprises treating thepopulation of cells with a compound (e.g., enzyme) that removes theselectable marker from a cell surface of at least one cell of the targetcell population. In some embodiments, the population of target cells istreated with the compound prior to the contacting of the target cellwith the stimulating compound. In some embodiments, the compound cleavesthe selectable marker from the cell surface of the at least one cell. Insome embodiments, the target cell is an endocrine cell. In someembodiments, the stimulating compound comprises glucose. In someembodiments, the endocrine cell is a β cell. In some embodiments, the βcell is an SC-β cell. In some embodiments, the stimulating compoundcomprises isoproterenol. In some embodiments, the endocrine cell is anEC cell. The method of claim 102, wherein the stimulating compound isglucose and the one or more cells is an EC cell. In some embodiments,the stimulating compound is isoproterenol and the one or more cells is aβ cell. In some embodiments, selecting the target cell separates thetarget cell from the one or more cells of the population of cells.

Irradiation

In some embodiments, the insulin producing endocrine cells (e.g., stemcell derived beta cells) can be cultured in the presence of a feederlayer of cells. Such cells may, for example, be of murine or humanorigin. The insulin producing endocrine cells (e.g., stem cell derivedbeta cells) can also be irradiated, chemically inactivated by treatmentwith a chemical inactivator such as mitomycin c, or otherwise treated toinhibit their proliferation if desired. In other embodiments, theinsulin producing endocrine cells (e.g., stem cell derived beta cells)are cultured without feeder cells.

The pluripotent stem cells can be maintained in an undifferentiatedstate even without feeder cells. The environment for feeder-freecultures includes a suitable culture substrate, particularly anextracellular matrix such as Matrigel® or laminin. Typically, enzymaticdigestion is halted before cells become completely dispersed (˜5 mM withcollagenase IV). Clumps of ˜10 to 2,000 cells are then plated directlyonto the substrate without further dispersal. Feeder-free cultures aresupported by a nutrient medium containing factors that supportproliferation of the cells without differentiation. Such factors may beintroduced into the medium by culturing the medium with cells secretingsuch factors, such as irradiated (˜4,000 rad) primary mouse embryonicfibroblasts, telomerized mouse fibroblasts, or fibroblast-like cellsderived from pPS cells. Medium can be conditioned by plating the feedersat a density of ˜5-6×104 cm⁻² in a serum free medium such as KO DMEMsupplemented with 20% serum replacement and 4 ng/mL bFGF. Medium thathas been conditioned for 1-2 days is supplemented with further bFGF, andused to support pluripotent stem cell culture for 1-2 days. Features ofthe feeder-free culture method are further discussed in InternationalPatent Publication WO 01/51616; and Xu et al., Nat. Biotechnol. 19:971,2001.

X. Pharmaceutical Compositions

The present disclosure relates to a therapeutic composition containingcells produced by any of the foregoing methods or containing any of theforegoing cell populations. The therapeutic compositions can furthercomprise a physiologically compatible solution including, for example,artificial cerebrospinal fluid or phosphate-buffered saline. Thetherapeutic composition can be used to treat, prevent, or stabilizediabetes. For example, somatic cells or stem cells can be obtained froman individual in need of treatment or from a healthy individual andreprogrammed to stem cell derived beta cells by the method of thepresent disclosure. In one embodiment of the present disclosure the stemcell derived beta cells are sorted and enriched and introduced into theindividual to treat the condition. In another embodiment the stem cellsare cultured under conditions suitable for differentiation into betacells prior to introduction into the individual, and can be used toreplace or assist the normal function of diseased or damaged tissue. Thegreat advantage of the present disclosure is that it provides anessentially limitless supply of patient specific human beta cells orcompatible stem cell derived beta cells from healthy individuals withthe same HLA type suitable for transplantation. The use of autologousand/or compatible cells in cell therapy offers a major advantage overthe use of non-autologous cells, which are likely to be subject toimmunological rejection. In contrast, autologous cells are unlikely toelicit significant immunological responses.

In some cases, the present disclosure provides pharmaceuticalcompositions that can utilize non-native pancreatic β cell (beta cells)populations and cell components and products in various methods fortreatment of a disease (e.g., diabetes). Certain cases encompasspharmaceutical compositions comprising live cells (e.g., non-nativepancreatic β cells alone or admixed with other cell types). Other casesencompass pharmaceutical compositions comprising non-native pancreatic βcell components (e.g., cell lysates, soluble cell fractions, conditionedmedium, ECM, or components of any of the foregoing) or products (e.g.,trophic and other biological factors produced by non-native pancreatic βcells or through genetic modification, conditioned medium fromnon-native pancreatic β cell culture). In either case, thepharmaceutical composition may further comprise other active agents,such as anti-inflammatory agents, exogenous small molecule agonists,exogenous small molecule antagonists, anti-apoptotic agents,antioxidants, and/or growth factors known to a person having skill inthe art.

Pharmaceutical compositions of the present disclosure can comprisenon-native pancreatic β cell, or components or products thereof,formulated with a pharmaceutically acceptable carrier (e.g. a medium oran excipient). The term pharmaceutically acceptable carrier (or medium),which may be used interchangeably with the term biologically compatiblecarrier or medium, can refer to reagents, cells, compounds, materials,compositions, and/or dosage forms that are not only compatible with thecells and other agents to be administered therapeutically, but also aresuitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or othercomplication. Suitable pharmaceutically acceptable carriers can includewater, salt solution (such as Ringer's solution), alcohols, oils,gelatins, and carbohydrates, such as lactose, amylose, or starch, fattyacid esters, hydroxymethylcellulose, and polyvinyl pyrolidine. Suchpreparations can be sterilized, and if desired, mixed with auxiliaryagents such as lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, andcoloring. Pharmaceutical compositions comprising cellular components orproducts, but not live cells, can be formulated as liquids.Pharmaceutical compositions comprising living non-native pancreatic βcells can be formulated as liquids, semisolids (e.g., gels, gelcapsules, or liposomes) or solids (e.g., matrices, scaffolds and thelike).

As used here, the term “pharmaceutically acceptable” can refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used here, the term “pharmaceutically-acceptable carrier” can referto a pharmaceutically-acceptable material, composition or vehicle, suchas a liquid or solid filler, diluent, excipient, manufacturing aid(e.g., lubricant, talc magnesium, calcium or zinc stearate, or stericacid), or solvent encapsulating material, involved in carrying ortransporting the subject compound from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. Some examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, methylcellulose,ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, suchas magnesium stearate, sodium lauryl sulfate and talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient,” “carrier,” “pharmaceutically acceptablecarrier” or the like are used interchangeably herein.

The phrase “therapeutically-effective amount” as used herein in respectto a population of cells means that amount of relevant cells in apopulation of cells, e.g., SC-β cells or mature pancreatic β cells, orcomposition comprising SC-β cells of the present disclosure which iseffective for producing some desired therapeutic effect in at least asub-population of cells in an animal at a reasonable benefit/risk ratioapplicable to any medical treatment. For example, an amount of apopulation of SC-β cells administered to a subject that is sufficient toproduce a statistically significant, measurable change in at least onesymptom of Type 1, Type 1.5 or Type 2 diabetes, such as glycosylatedhemoglobin level, fasting blood glucose level, hypoinsulinemia, etc.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. Generally, a therapeuticallyeffective amount can vary with the subject's history, age, condition,sex, as well as the severity and type of the medical condition in thesubject, and administration of other pharmaceutically active agents.

In some instances, pharmaceutical compositions of the stem cell derivedbeta cells are formulated in a conventional manner using one or morephysiologically acceptable carriers including excipients and auxiliarieswhich facilitate processing of the active compounds into preparationswhich can be used pharmaceutically. Proper formulation is dependent uponthe route of administration chosen. A summary of pharmaceuticalcompositions described herein is found, for example, in Remington: TheScience and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: MackPublishing Company, 1995); Hoover, John E., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. andLachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York,N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems,Seventh Ed. (Lippincott Williams & Wilkins 1999).

Pharmaceutical compositions are optionally manufactured in aconventional manner, such as, by way of example only, by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or compression processes.

In certain embodiments, compositions may also include one or more pHadjusting agents or buffering agents, including acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids; bases such assodium hydroxide, sodium phosphate, sodium borate, sodium citrate,sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; andbuffers such as citrate/dextrose, sodium bicarbonate and ammoniumchloride. Such acids, bases and buffers are included in an amountrequired to maintain pH of the composition in an acceptable range.

In other embodiments, compositions can also include one or more salts inan amount required to bring osmolality of the composition into anacceptable range. Such salts include those having sodium, potassium orammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

The pharmaceutical compositions described herein are administered by anysuitable administration route, including but not limited to, oral,parenteral (e.g., intravenous, subcutaneous, intramuscular,intracerebral, intracerebroventricular, intra-articular,intraperitoneal, or intracranial), intranasal, buccal, sublingual, orrectal administration routes. In some instances, the pharmaceuticalcomposition is formulated for parenteral (e.g., intravenous,subcutaneous, intramuscular, intracerebral, intracerebroventricular,intra-articular, intraperitoneal, or intracranial) administration.

The pharmaceutical compositions described herein are formulated into anysuitable dosage form, including but not limited to, aqueous oraldispersions, liquids, gels, syrups, elixirs, slurries, suspensions andthe like, for oral ingestion by an individual to be treated, solid oraldosage forms, aerosols, controlled release formulations, fast meltformulations, effervescent formulations, lyophilized formulations,tablets, powders, pills, dragees, capsules, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, multiparticulate formulations, and mixed immediate releaseand controlled release formulations. In some embodiments, thepharmaceutical compositions are formulated into capsules. In someembodiments, the pharmaceutical compositions are formulated intosolutions (for example, for IV administration). In some cases, thepharmaceutical composition is formulated as an infusion. In some cases,the pharmaceutical composition is formulated as an injection.

The pharmaceutical solid dosage forms described herein optionallyinclude a compound described herein and one or more pharmaceuticallyacceptable additives such as a compatible carrier, binder, fillingagent, suspending agent, flavoring agent, sweetening agent,disintegrating agent, dispersing agent, surfactant, lubricant, colorant,diluent, solubilizer, moistening agent, plasticizer, stabilizer,penetration enhancer, wetting agent, anti-foaming agent, antioxidant,preservative, or one or more combination thereof.

In still other aspects, using standard coating procedures, such as thosedescribed in Remington's Pharmaceutical Sciences, 20th Edition (2000), afilm coating is provided around the compositions. In some embodiments,the compositions are formulated into particles (for example foradministration by capsule) and some or all of the particles are coated.In some embodiments, the compositions are formulated into particles (forexample for administration by capsule) and some or all of the particlesare microencapsulated. In some embodiments, the compositions areformulated into particles (for example for administration by capsule)and some or all of the particles are not microencapsulated and areuncoated.

In certain embodiments, compositions provided herein may also includeone or more preservatives to inhibit microbial activity. Suitablepreservatives include mercury-containing substances such as merfen andthiomersal; stabilized chlorine dioxide; and quaternary ammoniumcompounds such as benzalkonium chloride, cetyltrimethylammonium bromideand cetylpyridinium chloride.

In some embodiments, a composition of the present disclosure cancomprise the stem cell derived beta cells, in an amount that iseffective to treat or prevent e.g., diabetes. A pharmaceuticalcomposition can comprise the stem cell derived beta cells as describedherein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions can comprise buffers such as neutral buffered saline,phosphate buffered saline and the like; carbohydrates such as glucose,mannose, sucrose or dextrans, mannitol; proteins; polypeptides or aminoacids such as glycine; antioxidants; chelating agents such as EDTA orglutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

Pharmaceutical compositions can comprise auxiliary components as wouldbe familiar to a person having skill in the art. For example, they cancontain antioxidants in ranges that vary depending on the kind ofantioxidant used. Reasonable ranges for commonly used antioxidants areabout 0.01% to about 0.15% weight by volume of EDTA, about 0.01% toabout 2.0% weight volume of sodium sulfite, and about 0.01% to about2.0% weight by volume of sodium metabisulfite. One skilled in the artmay use a concentration of about 0.1% weight by volume for each of theabove. Other representative compounds include mercaptopropionyl glycine,N-acetyl cysteine, β-mercaptoethylamine, glutathione and similarspecies, although other antioxidant agents suitable for renaladministration, e.g. ascorbic acid and its salts or sulfite or sodiummetabisulfite may also be employed.

A buffering agent can be used to maintain the pH of formulations in therange of about 4.0 to about 8.0; so as to minimize irritation in thetarget tissue. For direct intraperitoneal injection, formulations shouldbe at pH 7.2 to 7.5, preferably at pH 7.35-7.45. The compositions mayalso include tonicity agents suitable for administration to the kidney.Among those suitable is sodium chloride to make formulationsapproximately isotonic with blood.

In certain cases, pharmaceutical compositions are formulated withviscosity enhancing agents. Exemplary agents are hydroxyethylcellulose,hydroxypropylcellulose, methylcellulose, and polyvinylpyrrolidone. Thepharmaceutical compositions may have cosolvents added if needed.Suitable cosolvents may include glycerin, polyethylene glycol (PEG),polysorbate, propylene glycol, and polyvinyl alcohol. Preservatives mayalso be included, e.g., benzalkonium chloride, benzethonium chloride,chlorobutanol, phenylmercuric acetate or nitrate, thimerosal, or methylor propylparabens.

Pharmaceutical compositions comprising cells, cell components or cellproducts may be delivered to the kidney of a patient in one or more ofseveral methods of delivery known in the art. In some cases, thecompositions are delivered to the kidney (e.g., on the renal capsuleand/or underneath the renal capsule). In another embodiment, thecompositions may be delivered to various locations within the kidney viaperiodic intraperitoneal or intrarenal injection. Alternatively, thecompositions may be applied in other dosage forms known to those skilledin the art, such as pre-formed or in situ-formed gels or liposomes.

Pharmaceutical compositions comprising live cells in a semi-solid orsolid carrier are may be formulated for surgical implantation on orbeneath the renal capsule. It should be appreciated that liquidcompositions also may be administered by surgical procedures. Inparticular cases, semi-solid or solid pharmaceutical compositions maycomprise semi-permeable gels, lattices, cellular scaffolds and the like,which may be non-biodegradable or biodegradable. For example, in certaincases, it may be desirable or appropriate to sequester the exogenouscells from their surroundings, yet enable the cells to secrete anddeliver biological molecules (e.g., insulin) to surrounding cells or theblood stream. In these cases, cells may be formulated as autonomousimplants comprising living non-native pancreatic β cells or cellpopulation comprising non-native pancreatic β cell surrounded by anon-degradable, selectively permeable barrier that physically separatesthe transplanted cells from host tissue. Such implants are sometimesreferred to as “immunoprotective,” as they have the capacity to preventimmune cells and macromolecules from killing the transplanted cells inthe absence of pharmacologically induced immunosuppression.

In other cases, various degradable gels and networks can be used for thepharmaceutical compositions of the present disclosure. For example,degradable materials particularly suitable for sustained releaseformulations include biocompatible polymers, such as poly(lactic acid),poly (lactic-co-glycolic acid), methylcellulose, hyaluronic acid,collagen, and the like.

In other cases, it may be desirable or appropriate to deliver the cellson or in a biodegradable, preferably bioresorbable or bioabsorbable,scaffold or matrix. These typically three-dimensional biomaterialscontain the living cells attached to the scaffold, dispersed within thescaffold, or incorporated in an extracellular matrix entrapped in thescaffold. Once implanted into the target region of the body, theseimplants become integrated with the host tissue, wherein thetransplanted cells gradually become established.

Examples of scaffold or matrix (sometimes referred to collectively as“framework”) material that may be used in the present disclosure includenonwoven mats, porous foams, or self-assembling peptides. Nonwoven mats,for example, may be formed using fibers comprising a syntheticabsorbable copolymer of glycolic and lactic acids (PGA/PLA), foams,and/or poly(epsilon-caprolactone)/poly(glycolic acid) (PCL/PGA)copolymer.

In another embodiment, the framework is a felt, which can be composed ofa multifilament yarn made from a bioabsorbable material, e.g., PGA, PLA,PCL copolymers or blends, or hyaluronic acid. The yarn is made into afelt using standard textile processing techniques consisting ofcrimping, cutting, carding and needling. In another embodiment, cellsare seeded onto foam scaffolds that may be composite structures. In manyof the abovementioned cases, the framework may be molded into a usefulshape. Furthermore, it will be appreciated that non-native pancreatic βcells may be cultured on pre-formed, non-degradable surgical orimplantable devices.

The matrix, scaffold or device may be treated prior to inoculation ofcells in order to enhance cell attachment. For example, prior toinoculation, nylon matrices can be treated with 0.1 molar acetic acidand incubated in polylysine, PBS, and/or collagen to coat the nylon.Polystyrene can be similarly treated using sulfuric acid. The externalsurfaces of a framework may also be modified to improve the attachmentor growth of cells and differentiation of tissue, such as by plasmacoating the framework or addition of one or more proteins (e.g.,collagens, elastic fibers, reticular fibers), glycoproteins,glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate,chondroitin-6-sulfate, dermatan sulfate, keratin sulfate), a cellularmatrix, and/or other materials such as, but not limited to, gelatin,alginates, agar, agarose, and plant gums, among others.

In one aspect, the present disclosure provided devices comprising a cellcluster comprising at least one pancreatic β cell. A device providedherein can be configured to produce and release insulin when implantedinto a subject. A device can comprise a cell cluster comprising at leastone pancreatic β cell, e.g., a non-native pancreatic β cell. A cellcluster in the device can exhibit in vitro GSIS. A device can furthercomprise a semipermeable membrane. The semipermeable membrane can beconfigured to retain the cell cluster in the device and permit passageof insulin secreted by the cell cluster. In some cases of the device,the cell cluster can be encapsulated by the semipermeable membrane. Theencapsulation can be performed by any technique available to one skilledin the art. The semipermeable membrane can also be made of any suitablematerial as one skilled in the art would appreciate and verify. Forexample, the semipermeable membrane can be made of polysaccharide orpolycation. In some cases, the semipermeable membrane can be made ofpoly(lactide) (PLA), poly(glycolic acid) (PGA),poly(lactide-co-glycolide) (PLGA), and other polyhydroxyacids,poly(caprolactone), polycarbonates, polyamides, polyanhydrides,polyphosphazene, polyamino acids, polyortho esters, polyacetals,polycyanoacrylates, biodegradable polyurethanes, albumin, collagen,fibrin, polyamino acids, prolamines, alginate, agarose, agarose withgelatin, dextran, polyacrylates, ethylene-vinyl acetate polymers andother acyl-substituted cellulose acetates and derivatives thereof,polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride,poly(vinyl imidazole), chlorosulphonated polyolefins, polyethyleneoxide, or any combinations thereof. In some cases, the semipermeablemembrane comprises alginate. In some cases, the cell cluster isencapsulated in a microcapsule that comprises an alginate coresurrounded by the semipermeable membrane. In some cases, the alginatecore is modified, for example, to produce a scaffold comprising analginate core having covalently conjugated oligopeptides with an RGDsequence (arginine, glycine, aspartic acid). In some cases, the alginatecore is modified, for example, to produce a covalently reinforcedmicrocapsule having a chemoenzymatically engineered alginate of enhancedstability. In some cases, the alginate core is modified, for example, toproduce membrane-mimetic films assembled by in-situ polymerization ofacrylate functionalized phospholipids. In some cases, microcapsules arecomposed of enzymatically modified alginates using epimerases, In somecases, microcapsules comprise covalent links between adjacent layers ofthe microcapsule membrane. In some embodiment, the microcapsulecomprises a subsieve-size capsule comprising alginate coupled withphenol moieties. In some cases, the microcapsule comprises a scaffoldcomprising alginate-agarose. In some cases, the SC-β cell is modifiedwith PEG before being encapsulated within alginate. In some cases, theisolated populations of cells, e.g., SC-β cells are encapsulated inphotoreactive liposomes and alginate. It should be appreciated that thealginate employed in the microcapsules can be replaced with othersuitable biomaterials, including, without limitation, polyethyleneglycol (PEG), chitosan, polyester hollow fibers, collagen, hyaluronicacid, dextran with ROD, BHD and polyethylene glycol-diacrylate (PEGDA),poly(MPC-co-n-butyl methacrylate-co-4-vinylphenyl boronic acid) (PMBV)and poly(vinyl alcohol) (PVA), agarose, agarose with gelatin, andmultilayer cases of these. In some cases, the device provided hereincomprise extracorporeal segment, e.g., part of the device can be outsidea subject's body when the device is implanted in the subject. Theextracorporeal segment can comprise any functional component of thedevice, with or without the cells or cell cluster provided herein.

XI. Methods of Treating

Further provided herein are methods for treating or preventing a diseasein a subject. A composition comprising the cell clusters or cellsprovided herein or generated according to the methods provided hereincan be administered into a subject to restore a degree of pancreaticfunction in the subject. For example, the cell clusters resemblingendogenous pancreatic islets, or the cells resembling endogenouspancreatic β cells (e.g., non-native pancreatic β cells or SC-β cells)or the precursors thereof can be transplanted to a subject to treatdiabetes.

The methods can comprise transplanting the cell cluster or the celldisclosed in the application to a subject, e.g., a subject in needthereof. The term “transplanting” can refer to the placement of cells orcell clusters, any portion of the cells or cell clusters thereof, or anycompositions comprising cells, cell clusters or any portion thereof,into a subject, by a method or route which results in at least partiallocalization of the introduced cells or cell clusters at a desired site.The cells or cell clusters can be implanted directly to the pancreas, oralternatively be administered by any appropriate route which results indelivery to a desired location in the subject where at least a portionof the implanted cells or cell remain viable. The period of viability ofthe cells or cell clusters after administration to a subject can be asshort as a few hours, e.g. twenty-four hours, to a few days, to as longas several years. In some instances, the cells or cell clusters, or anyportion of the cells or cell clusters thereof, can also betransadministered at a non-pancreatic location, such as in the liver orsubcutaneously, for example, in a capsule (e.g., microcapsule) tomaintain the implanted cells or cell clusters at the implant locationand avoid migration.

As used herein, the term “treating” and “treatment” can refer toadministering to a subject an effective amount of a composition (e.g.,cell clusters or a portion thereof) so that the subject as a reductionin at least one symptom of the disease or an improvement in the disease,for example, beneficial or desired clinical results. For purposes ofthis disclosure, beneficial or desired clinical results include, but arenot limited to, alleviation of one or more symptoms, diminishment ofextent of disease, stabilized (e.g., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (e.g., partial or total), whetherdetectable or undetectable. Treating can refer to prolonging survival ascompared to expected survival if not receiving treatment. Thus, one ofskill in the art realizes that a treatment may improve the diseasecondition, but may not be a complete cure for the disease. As usedherein, the term “treatment” includes prophylaxis.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. Inpreferred embodiments, the compositions are administered by intravenousinfusion or injection.

By “treatment,” “prevention” or “amelioration” of a disease or disorderis meant delaying or preventing the onset of such a disease or disorder,reversing, alleviating, ameliorating, inhibiting, slowing down orstopping the progression, aggravation or deterioration the progressionor severity of a condition associated with such a disease or disorder.In one embodiment, the symptoms of a disease or disorder are alleviatedby at least 5%, at least 10%, at least 20%, at least 30%, at least 40%,or at least 50%.

Treatment of Diabetes is determined by standard medical methods. A goalof Diabetes treatment is to bring sugar levels down to as close tonormal as is safely possible. Commonly set goals are 80-120 milligramsper deciliter (mg/dl) before meals and 100-140 mg/dl at bedtime. Aparticular physician may set different targets for the patent, dependingon other factors, such as how often the patient has low blood sugarreactions. Useful medical tests include tests on the patient's blood andurine to determine blood sugar level, tests for glycosylated hemoglobinlevel (HbA1c; a measure of average blood glucose levels over the past2-3 months, normal range being 4-6%), tests for cholesterol and fatlevels, and tests for urine protein level. Such tests are standard testsknown to those of skill in the art (see, for example, American DiabetesAssociation, 1998). A successful treatment program can also bedetermined by having fewer patients in the program with complicationsrelating to Diabetes, such as diseases of the eye, kidney disease, ornerve disease.

Delaying the onset of diabetes in a subject refers to delay of onset ofat least one symptom of diabetes, e.g., hyperglycemia, hypoinsulinemia,diabetic retinopathy, diabetic nephropathy, blindness, memory loss,renal failure, cardiovascular disease (including coronary arterydisease, peripheral artery disease, cerebrovascular disease,atherosclerosis, and hypertension), neuropathy, autonomic dysfunction,hyperglycemic hyperosmolar coma, or combinations thereof, for at least 1week, at least 2 weeks, at least 1 month, at least 2 months, at least 6months, at least 1 year, at least 2 years, at least 5 years, at least 10years, at least 20 years, at least 30 years, at least 40 years or more,and can include the entire lifespan of the subject.

In some aspects, the disclosure relates to a method comprisingimplanting in a subject a device comprising a cell or cell clusterprovided herein (e.g., insulin producing cells), wherein the devicereleases insulin in an amount sufficient for a reduction of bloodglucose levels in the subject. In some embodiments, the insulinproducing cells are glucose responsive insulin producing cells.

In some embodiments, the reduction of blood glucose levels in thesubject, as induced by the transplantation of the cell or cell cluster,or the device provided herein, results in an amount of glucose which islower than the diabetes threshold. In some embodiments, the subject is amammalian subject. In some embodiments, the mammalian subject is human.In some embodiments, the amount of glucose is reduced to lower than thediabetes threshold in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after theimplanting.

As described in detail above, the pharmaceutical compositions of thepresent disclosure can be specially formulated for administration insolid or liquid form, including those adapted for the following: (1)oral administration, for example, drenches (aqueous or non-aqueoussolutions or suspensions), lozenges, dragees, capsules, pills, tablets(e.g., those targeted for buccal, sublingual, and systemic absorption),boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; (3) topical application,for example, as a cream, ointment, or a controlled-release patch orspray applied to the skin; (4) intravaginally or intrarectally, forexample, as a pessary, cream or foam; (5) sublingually; (6) ocularly;(7) transdermally; (8) transmucosally; or (9) nasally. Additionally,compounds can be implanted into a patient or injected using a drugdelivery system. See, for example, Urquhart, et al., Ann. Rev.Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. “Controlled Releaseof Pesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S.Pat. No. 3,773,919; and 35 U.S. Pat. No. 3,270,960.

A subject that can be treated by the methods herein can be a human or anon-human animal. In some cases, a subject can be a mammal. Examples ofa subject include but are not limited to primates, e.g., a monkey, achimpanzee, a bamboo, or a human. In some cases, a subject is a human. Asubject can be non-primate animals, including, but not limited to, adog, a cat, a horse, a cow, a pig, a sheep, a goat, a rabbit, and thelike. In some cases, a subject receiving the treatment is a subject inneed thereof, e.g., a human in need thereof.

In certain embodiments, the subject is a mammal, e.g., a primate, e.g.,a human. The terms, “patient” and “subject” are used interchangeablyherein. Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of Type 1diabetes, Type 2 Diabetes Mellitus, or pre-diabetic conditions. Inaddition, the methods described herein can be used to treat domesticatedanimals and/or pets. A subject can be male or female. A subject can beone who has been previously diagnosed with or identified as sufferingfrom or having Diabetes (e.g., Type 1 or Type 2), one or morecomplications related to Diabetes, or a pre-diabetic condition, andoptionally, but need not have already undergone treatment for theDiabetes, the one or more complications related to Diabetes, or thepre-diabetic condition. A subject can also be one who is not sufferingfrom Diabetes or a pre-diabetic condition. A subject can also be one whohas been diagnosed with or identified as suffering from Diabetes, one ormore complications related to Diabetes, or a pre-diabetic condition, butwho show improvements in known Diabetes risk factors as a result ofreceiving one or more treatments for Diabetes, one or more complicationsrelated to Diabetes, or the pre-diabetic condition. Alternatively, asubject can also be one who has not been previously diagnosed as havingDiabetes, one or more complications related to Diabetes, or apre-diabetic condition. For example, a subject can be one who exhibitsone or more risk factors for Diabetes, complications related toDiabetes, or a pre-diabetic condition, or a subject who does not exhibitDiabetes risk factors, or a subject who is asymptomatic for Diabetes,one or more Diabetes-related complications, or a pre-diabetic condition.A subject can also be one who is suffering from or at risk of developingDiabetes or a pre-diabetic condition. A subject can also be one who hasbeen diagnosed with or identified as having one or more complicationsrelated to Diabetes or a pre-diabetic condition as defined herein, oralternatively, a subject can be one who has not been previouslydiagnosed with or identified as having one or more complications relatedto Diabetes or a pre-diabetic condition.

The methods can comprise transplanting the cell cluster to a subjectusing any means in the art. For example the methods can comprisetransplanting the cell cluster via the intraperitoneal space, renalsubcapsule, renal capsule, omentum, subcutaneous space, or viapancreatic bed infusion. For example, transplanting can be subcapsulartransplanting, intramuscular transplanting, or intraportaltransplanting, e.g., intraportal infusion. Immunoprotectiveencapsulation can be implemented to provide immunoprotection to the cellclusters. In some cases, the methods of treatment provided herein cancomprise administer immune response modulator for modulating or reducingtransplant rejection response or other immune response against theimplant (e.g., the cells or the device). Examples of immune responsemodulator that can be used in the methods can include purine synthesisinhibitors like Azathioprine and Mycophenolic acid, pyrimidine synthesisinhibitors like Leflunomide and Teriflunomide, antifolate likeMethotrexate, Tacrolimus, Ciclosporin, Pimecrolimus, Abetimus,Gusperimus, Lenalidomide, Pomalidomide, Thalidomide, PDE4 inhibitor,Apremilast, Anakinra, Sirolimus, Everolimus, Ridaforolimus,Temsirolimus, Umirolimus, Zotarolimus, Anti-thymocyte globulinantibodies, Anti-lymphocyte globulin antibodies, CTLA-4, fragmentthereof, and fusion proteins thereof like Abatacept and Belatacept, TNFinhibitor like Etanercept and Pegsunercept, Aflibercept, Alefacept,Rilonacept, antibodies against complement component 5 like Eculizumab,anti-TNF antibodies like Adalimumab, Afelimomab, Certolizumab pegol,Golimumab, Infliximab, and Nerelimomab, antibodies against Interleukin 5like Mepolizumab, anti-Ig E antibodies like Omalizumab, anti-Interferonantibodies like Faralimomab, anti-IL-6 antibodies like Elsilimomab,antibodies against IL-12 and IL-23 like Lebrikizumab and Ustekinumab,anti-IL-17A antibodies like Secukinumab, anti-CD3 antibodies likeMuromonab-CD3, Otelixizumab, Teplizumab, and Visilizumab, anti-CD4antibodies like Clenoliximab, Keliximab, and Zanolimumab, anti-CD11aantibodies like Efalizumab, anti-CD18 antibodies like Erlizumab,anti-CD20 antibodies like Obinutuzumab, Rituximab, Ocrelizumab andPascolizumab, anti-CD23 antibodies like Gomiliximab and Lumiliximab,anti-CD40 antibodies like Teneliximab and Toralizumab, antibodiesagainst CD62L/L-selectin like Aselizumab, anti-CD80 antibodies likeGaliximab, anti-CD147/Basigin antibodies like Gavilimomab, anti-CD154antibodies like Ruplizumab, anti-BLyS antibodies like Belimumab andBlisibimod, anti-CTLA-4 antibodies like Ipilimumab and Tremelimumab,anti-CAT antibodies like Bertilimumab, Lerdelimumab, and Metelimumab,anti-Integrin antibodies like Natalizumab, antibodies againstInterleukin-6 receptor like Tocilizumab, anti-LFA-1 antibodies likeOdulimomab, antibodies against IL-2 receptor/CD25 like Basiliximab,Daclizumab, and Inolimomab, antibodies against T-lymphocyte (Zolimomabaritox) like Atorolimumab, Cedelizumab, Fontolizumab, Maslimomab,Morolimumab, Pexelizumab, Reslizumab, Rovelizumab, Siplizumab,Talizumab, Telimomab aritox, Vapaliximab, and Vepalimomab.

“Antifoaming agents” reduce foaming during processing which can resultin coagulation of aqueous dispersions, bubbles in the finished film, orgenerally impair processing. Exemplary anti-foaming agents includesilicon emulsions or sorbitan sesquoleate.

“Antioxidants” include, for example, butylated hydroxytoluene (BHT),sodium ascorbate, ascorbic acid, sodium metabisulfite and tocopherol. Incertain embodiments, antioxidants enhance chemical stability whererequired.

Formulations described herein may benefit from antioxidants, metalchelating agents, thiol containing compounds and other generalstabilizing agents. Examples of such stabilizing agents, include, butare not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/vmonothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% toabout 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h) arginine, (i)heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosanpolysulfate and other heparinoids, (m) divalent cations such asmagnesium and zinc; or (n) combinations thereof.

“Binders” impart cohesive qualities and include, e.g., alginic acid andsalts thereof; cellulose derivatives such as carboxymethylcellulose,methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose,hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel®),ethylcellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g.,Avicel®); microcrystalline dextrose; amylose; magnesium aluminumsilicate; polysaccharide acids; bentonites; gelatin;polyvinylpyrrolidone/vinyl acetate copolymer; crospovidone; povidone;starch; pregelatinized starch; tragacanth, dextrin, a sugar, such assucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol,xylitol (e.g., Xylitab®), and lactose; a natural or synthetic gum suchas acacia, tragacanth, ghatti gum, mucilage of isapol husks,polyvinylpyrrolidone (e.g., Polyvidone® CL, Kollidon® CL, Polyplasdone®XL-10), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodiumalginate, and the like.

A “carrier” or “carrier materials” include any commonly used excipientsin pharmaceutics and should be selected on the basis of compatibilitywith compounds disclosed herein, such as, compounds of ibrutinib and Ananticancer agent, and the release profile properties of the desireddosage form. Exemplary carrier materials include, e.g., binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, diluents, and thelike. “Pharmaceutically compatible carrier materials” may include, butare not limited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like. See,e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed(Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999).

“Dispersing agents,” and/or “viscosity modulating agents” includematerials that control the diffusion and homogeneity of a drug throughliquid media or a granulation method or blend method. In someembodiments, these agents also facilitate the effectiveness of a coatingor eroding matrix. Exemplary diffusion facilitators/dispersing agentsinclude, e.g., hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG,polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and thecarbohydrate-based dispersing agents such as, for example, hydroxypropylcelluloses (e.g., HPC, HPC-SL, and HPC-L), hydroxypropylmethylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M),carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), noncrystallinecellulose, magnesium aluminum silicate, triethanolamine, polyvinylalcohol (PVA), vinyl pyrrolidone/vinyl acetate copolymer (S630),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol), poloxamers (e.g., PluronicsF68®, F88®, and F108®, which are block copolymers of ethylene oxide andpropylene oxide); and poloxamines (e.g., Tetronic 908®, also known asPoloxamine 908®, which is a tetrafunctional block copolymer derived fromsequential addition of propylene oxide and ethylene oxide toethylenediamine (BASF Corporation, Parsippany, N.J.)),polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetatecopolymer (S-630), polyethylene glycol, e.g., the polyethylene glycolcan have a molecular weight of about 300 to about 6000, or about 3350 toabout 4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g.,gum tragacanth and gum acacia, guar gum, xanthans, including xanthangum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose,methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodiumalginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitanmonolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates,chitosans and combinations thereof. Plasticizers such as cellulose ortriethyl cellulose can also be used as dispersing agents. Dispersingagents particularly useful in liposomal dispersions and self-emulsifyingdispersions are dimyristoyl phosphatidyl choline, natural phosphatidylcholine from eggs, natural phosphatidyl glycerol from eggs, cholesteroland isopropyl myristate.

Combinations of one or more erosion facilitator with one or morediffusion facilitator can also be used in the present compositions.

The term “diluent” refers to chemical compounds that are used to dilutethe compound of interest prior to delivery. Diluents can also be used tostabilize compounds because they can provide a more stable environment.Salts dissolved in buffered solutions (which also can provide pH controlor maintenance) are utilized as diluents in the art, including, but notlimited to a phosphate buffered saline solution. In certain embodiments,diluents increase bulk of the composition to facilitate compression orcreate sufficient bulk for homogenous blend for capsule filling. Suchcompounds include e.g., lactose, starch, mannitol, sorbitol, dextrose,microcrystalline cellulose such as Avicel®; dibasic calcium phosphate,dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate;anhydrous lactose, spray-dried lactose; pregelatinized starch,compressible sugar, such as Di-Pac® (Amstar); mannitol,hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetatestearate, sucrose-based diluents, confectioner's sugar; monobasiccalcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactatetrihydrate, dextrates; hydrolyzed cereal solids, amylose; powderedcellulose, calcium carbonate; glycine, kaolin; mannitol, sodiumchloride; inositol, bentonite, and the like.

“Filling agents” include compounds such as lactose, calcium carbonate,calcium phosphate, dibasic calcium phosphate, calcium sulfate,microcrystalline cellulose, cellulose powder, dextrose, dextrates,dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol,mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

“Lubricants” and “glidants” are compounds that prevent, reduce orinhibit adhesion or friction of materials. Exemplary lubricants include,e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, ahydrocarbon such as mineral oil, or hydrogenated vegetable oil such ashydrogenated soybean oil (Sterotex®), higher fatty acids and theiralkali-metal and alkaline earth metal salts, such as aluminum, calcium,magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes,Stearowet®, boric acid, sodium benzoate, sodium acetate, sodiumchloride, leucine, a polyethylene glycol (e.g., PEG-4000) or amethoxypolyethylene glycol such as Carbowax™, sodium oleate, sodiumbenzoate, glyceryl behenate, polyethylene glycol, magnesium or sodiumlauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil®, a starchsuch as corn starch, silicone oil, a surfactant, and the like.

“Plasticizers” are compounds used to soften the microencapsulationmaterial or film coatings to make them less brittle. Suitableplasticizers include, e.g., polyethylene glycols such as PEG 300, PEG400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propyleneglycol, oleic acid, triethyl cellulose and triacetin. In someembodiments, plasticizers can also function as dispersing agents orwetting agents.

“Solubilizers” include compounds such as triacetin, triethylcitrate,ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate,vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone,N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropylalcohol, cholesterol, bile salts, polyethylene glycol 200-600,glycofurol, transcutol, propylene glycol, and dimethyl isosorbide andthe like.

“Stabilizers” include compounds such as any antioxidation agents,buffers, acids, preservatives and the like.

“Suspending agents” include compounds such as polyvinylpyrrolidone,e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17,polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g.,the polyethylene glycol can have a molecular weight of about 300 toabout 6000, or about 3350 to about 4000, or about 7000 to about 5400,sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate,polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as,e.g., gum tragacanth and gum acacia, guar gum, xanthans, includingxanthan gum, sugars, cellulosics, such as, e.g., sodiumcarboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80,sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylatedsorbitan monolaurate, povidone and the like.

“Surfactants” include compounds such as sodium lauryl sulfate, sodiumdocusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitanmonooleate, polyoxyethylene sorbitan monooleate, polysorbates,polaxomers, bile salts, glyceryl monostearate, copolymers of ethyleneoxide and propylene oxide, e.g., Pluronic® (BASF), and the like. Someother surfactants include polyoxyethylene fatty acid glycerides andvegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40. In some embodiments, surfactants may be included toenhance physical stability or for other purposes.

“Viscosity enhancing agents” include, e.g., methyl cellulose, xanthangum, carboxymethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetatestearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinylalcohol, alginates, acacia, chitosans and combinations thereof.

“Wetting agents” include compounds such as oleic acid, glycerylmonostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamineoleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate,sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium saltsand the like.

The disclosure is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

Exemplary Embodiments

[1] A method comprising:contacting a population of pancreatic progenitor cells or precursorsthereof with a composition comprising at least one epigenetic modifyingcompound, wherein said contacting results in a population of endocrinecells with a reduced proportion of cells expressing VMAT or Cdx2 ascompared to a corresponding population of endocrine cells which is notcontacted with said at least one epigenetic modifying compound.[2] The method of paragraph [1], wherein said at least one epigeneticmodifying compound comprises one or more of a DNA methylation inhibitor,a histone acetyltransferase inhibitor, a histone deacetylase inhibitor,a histone methyltransferase inhibitor, or a bromodomain inhibitor.[3] The method of paragraph [2], wherein said at least one epigeneticmodifying compound comprises a histone methyltransferase inhibitor.[4] The method of paragraph [3], wherein said histone methyltransferaseinhibitor is an EZH2 inhibitor.[5] The method of paragraph [3] or [4], wherein said histonemethyltransferase inhibitor is at least one of DZNep, GSK126, orEPZ6438.[6] The method of paragraph [5], wherein said histone methyltransferaseinhibitor is DZNep.[7] The method of paragraph [6], wherein a concentration of said DZNepin said composition is greater than 0.1 μM.[8] The method of paragraph [7], wherein said concentration of saidDZNep is at least 0.5 μM.[9] The method of paragraph [7], wherein said concentration of saidDZNep is about 1 μM.[10] The method of any one of paragraphs [1] to [9], wherein said atleast one epigenetic modifying compound comprises a histone deacetylase(HDAC) inhibitor.[11] The method of paragraph [10], wherein said HDAC inhibitor is aClass I HDAC inhibitor, a Class II HDAC inhibitor, or a combinationthereof.[12] The method of paragraph [11], wherein said HDAC inhibitor is atleast one of KD5170, MC1568, or TMP195.[13] The method of paragraph [12], wherein said HDAC inhibitor isKD5170.[14] The method of paragraph [1], wherein said at least one epigeneticmodifying compound comprises an HDAC inhibitor and an EZH2 inhibitor.[15] The method of paragraph [1], wherein said at least one epigeneticmodifying compound comprises DZNep and KD5170.[16] The method of any one of paragraphs [1]-[15], wherein at least oneof said cells expressing VMAT is INS⁻.[17] The method of any one of paragraphs [1]-[16], wherein at least somecells of said population of pancreatic progenitor cells differentiateinto a population of PH cells.[18] The method of paragraph [17], wherein an increased proportion ofcells of said population of endocrine cells are NKX6.1⁻ or ChromA⁺ ascompared to said corresponding population of endocrine cells which isnot contacted with said at least one epigenetic modifying compound.[19] The method of paragraph [18], wherein at least one cell of saidincreased proportion of cells is NKX6.1⁻ and ChromA⁺.[20] The method of any one of paragraphs [1]-[19], wherein at least somecells of said population of pancreatic progenitor cells differentiateinto a population of β cells.[21] The method of paragraph [20], wherein said β cells are stem-cellderived β (SC-β) cells.[22] The method of paragraph [20] or [21], wherein said β cells expressC-PEP and NKX6-1.[23] The method of any one of paragraphs [20]-[22], wherein said β cellsexhibit an in vitro glucose-stimulated insulin secretion response to aglucose challenge.[24] The method of any one of paragraphs [1]-[23], wherein said methodis performed in vitro.[25] The method of paragraph [24], wherein said composition comprises atleast one of betacellulin, thiazovinin, retinoic acid, SANT1, XXI, Alk5iII, GC-1, LDN or staurosporine.[26] The method of paragraph [24] or [25], wherein said contacting isfor at least three days.[27] The method of paragraph [24] or [25], wherein said contacting isfor at least five days.[28] The method of paragraph [24] or [25], wherein said contacting isfor about seven days.[29] The method of any one of paragraphs [1]-[28], wherein at least onepancreatic progenitor cell of said population of pancreatic progenitorcells expresses at least one of PDX1 and NKX6-1.[30] The method of any one of paragraphs [1]-[29], wherein at least oneendocrine cell of said population of endocrine cells expresses CHGA.[31] A cell produced by the method of any one of paragraphs [1]-[30].[32] A composition that comprises a pancreatic progenitor cell, ahistone deacetylase (HDAC) inhibitor, a histone methyltransferaseinhibitor and optionally an endocrine cell.[33] The composition of paragraph [32], wherein said HDAC inhibitor is aClass I HDAC inhibitor, a Class II HDAC inhibitor, or a combinationthereof.[34] The composition of paragraph [33], wherein said HDAC inhibitor isat least one of KD5170, MC1568, or TMP195.[35] The composition of paragraph [34], wherein said HDAC inhibitor isKD5170.[36] The composition of paragraph [35], wherein a concentration of saidKD5170 in said composition is at least 0.1 μM.[37] The composition of paragraph [36], wherein said concentration ofsaid KD5170 is at least 0.5 μM.[38] The composition of paragraph [37], wherein said concentration ofsaid KD5170 is about [1 μM.[39] The composition of any one of paragraphs [32]-[38], wherein saidhistone methyltransferase inhibitor is an EZH2 inhibitor.[40] The composition of paragraph [39], wherein said histonemethyltransferase inhibitor is at least one of DZNep, GSK126, orEPZ6438.[41] The composition of paragraph [40], wherein said histonemethyltransferase inhibitor is DZNep.[42] The composition of paragraph [41], wherein a concentration of saidDZNep is at least 0.1 μM.[43] The composition of paragraph [42], wherein said concentration ofsaid DZNep is at least 0.5 μM.[44] The composition of paragraph [42], wherein said concentration ofsaid DZNep is about [1 μM.[45] The composition of paragraph [32], wherein said HDAC inhibitor isKD5170 and said histone methyltransferase inhibitor is DZNep.[46] The composition of any one of paragraphs [32]-[45], wherein saidcomposition is an in vitro composition.[47] The composition of paragraph [46], wherein said composition furthercomprises at least one of betacellulin, thiazovinin, retinoic acid,SANT1, XXI, Alk5i II, GC-1, LDN or staurosporine.[48] A method comprising contacting a pancreatic progenitor cell orprecursor thereof with a histone deacetylase (HDAC) inhibitor and ahistone methyltransferase inhibitor, wherein said contacting inducesdifferentiation of said pancreatic progenitor cell.[49] The method of paragraph [48], wherein said pancreatic progenitorcell differentiates into a β cell.[50] The method of paragraph [49], wherein said β cell is a stem-cellderived β (SC-β) cell.[51] The method of paragraph [49] or [50], wherein said β cell expressesC-PEP and NKX6-1.[52] The method of any one of paragraphs [49]-[51], wherein said β cellsexhibit an in vitro glucose-stimulated insulin secretion response to aglucose challenge.[53] The method of any one of paragraphs [48]-[52], wherein said HDACinhibitor is a Class I HDAC inhibitor, a Class II HDAC inhibitor, or acombination thereof.[54] The method of paragraph [53], wherein said HDAC inhibitor is atleast one of KD5170, MC1568, or TMP195.[55] The method of paragraph [54], wherein said HDAC inhibitor isKD5170.[56] The method of any one of paragraphs [48]-[55], wherein said histonemethyltransferase inhibitor is an EZH2 inhibitor.[57] The method of paragraph [56], wherein said histonemethyltransferase inhibitor is at least one of DZNep, GSK126, orEPZ6438.[58] The method of paragraph [57], wherein said histonemethyltransferase inhibitor is DZNep.[59] The method of paragraph [48], wherein said HDAC inhibitor is KD5170and said histone methyltransferase inhibitor is DZNep.[60] The method of any one of paragraphs [48]-[59], wherein said methodis performed in vitro.[61] A method comprising contacting a pancreatic progenitor cell orprecursor thereof with KD5170 in an amount sufficient to result indifferentiation of said cell.[62] The method of paragraph [61], further comprising contracting saidpancreatic progenitor cell with a histone methyltransferase inhibitor.[63] The method of paragraph [62], wherein said histonemethyltransferase inhibitor is at least one of DZNep, GSK126, orEPZ6438.[64] The method of paragraph [63], wherein said histonemethyltransferase inhibitor is DZNep.[65] The method of any one of paragraphs [61]-[64], wherein saidpancreatic progenitor cell differentiates into an endocrine cell.[66] The method of any one of paragraphs [61]-[65], wherein saidpancreatic progenitor cell differentiates into a β cell.[67] The method of paragraph [66], wherein said β cell is a stem-cellderived β (SC-β) cell.[68] The method of paragraph [66] or [67], wherein said β cell expressesC-PEP and NKX6-1.[69] The method of any one of paragraphs [66]-[68], wherein said β cellexhibits an in vitro glucose-stimulated insulin secretion response to aglucose challenge.[70] A method comprising:

-   -   a) differentiating a plurality of stem cells in vitro to obtain        a cell population comprising pancreatic progenitor cells or        precursors thereof;    -   b) contacting in vitro said cell population with a histone        deacetylase (HDAC) inhibitor to generate at least one endocrine        cell; and    -   c) maturing said endocrine cell in vitro to obtain at least one        SC-β cell.        [71] The method of paragraph [70], wherein said stem cells are        human pluripotent stem cells.        [72] The method of paragraph [70] or [71], further comprising        contacting said cell population with at least one of        betacellulin, thiazovinin, retinoic acid, SANT1, XXI, Alk5i II,        GC-1, LDN or staurosporine.        [7] The method of any one of paragraphs [70]-[72], wherein said        SC-β cell expresses C-PEP and NKX6-1.        [74] The method of any one of paragraphs [70]-[73], wherein said        SC-β cell exhibits an in vitro glucose-stimulated insulin        secretion response to a glucose challenge.        [75] The method of any one of paragraphs [70]-[74], further        comprising contracting said cell population with a histone        methyltransferase inhibitor.        [76] The method of paragraph [75], wherein said histone        methyltransferase inhibitor is at least one of DZNep, GSK126, or        EPZ6438.        [77] The method of paragraph [76], wherein said histone        methyltransferase inhibitor is DZNep.        [78] The method of any one of paragraphs [70]-[77], wherein said        HDAC inhibitor is KD5170.        [79] A method comprising contacting a cell population comprising        pancreatic progenitor cells or precursors thereof with a histone        methyltransferase inhibitor in vitro in an amount sufficient to        generate endocrine cells; and    -   maturing said endocrine cells in vitro to obtain at least one        SC-β cell that exhibits an in vitro glucose-stimulated insulin        secretion response to a glucose challenge.        [80] The method of paragraph [79], further comprising        differentiating a plurality of stem cells in vitro to obtain        said cell population comprising said pancreatic progenitor cells        or precursors thereof.        [81] The method of paragraph [79] or [80], further comprising        contacting said cell population with at least one of        betacellulin, thiazovinin, retinoic acid, SANT1, XXI, Alk5i II,        GC-1, LDN or staurosporine.        [82] The method of any one of paragraphs [79]-[81], further        comprising contacting said cell population with a histone        deacetylase (HDAC) inhibitor.        [83] The method of paragraph [82], wherein said HDAC inhibitor        is KD5170.        [84] The method of any one of paragraphs [79]-[83], wherein said        histone methyltransferase inhibitor is at least one of DZNep,        GSK126, or EPZ6438.        [85] The method of paragraph [84], wherein said histone        methyltransferase inhibitor is DZNep.        [86] A method for selecting a target cell from a population of        cells comprising:    -   (i) contacting said target cell with a stimulating compound,        wherein said contacting induces a selectable marker of said        target cell to localize to a cell surface of said target cell;        and    -   (ii) selecting said target cell based on said localization of        said selectable marker at said cell surface.        [87] The method of paragraph [86], wherein said selectable        marker comprises PSA-NCAM.        [88] The method of paragraph [87], wherein said selecting said        target cell is by cell sorting.        [89] The method of paragraph [88], wherein said selecting        comprises contacting said selectable marker of said target cell        with an antigen binding polypeptide when said selectable marker        is localized to said surface of said target cell.        [90] The method of paragraph [89], wherein said antigen binding        polypeptide comprises an antibody.        [91] The method of paragraph [90], wherein said antigen binding        polypeptide binds to said PSA-NCAM.        [92] The method of any one of paragraphs [86]-[91], wherein said        method further comprises treating said population of cells with        a compound that removes said selectable marker from a cell        surface of at least one cell of said population of cells.        [93] The method of paragraph [92], wherein said population of        cells is treated with said compound prior to said contacting        said target cell with said stimulating compound.        [94] The method of paragraph [92] or [93], wherein said compound        cleaves said selectable marker from said cell surface of said at        least one cell.        [95] The method of paragraph [94], wherein said compound is an        enzyme.        [96] The method of paragraph [95], wherein said compound is an        endosialidase.        [97] The method of paragraph [96], wherein said endosialidase is        endoneuraminidase (Endo-N).        [98] The method of any one of paragraphs [86]-[97], wherein said        target cell is an endocrine cell.        [99] The method of any one of paragraphs [86]-[98], wherein said        stimulating compound comprises at least one of arginine or        glucose.        [100] The method of paragraph [98], wherein said endocrine cell        is a β cell.        [101] The method of paragraph [101], wherein said β cell is an        SC-β cell.        [102] The method of paragraph [98], wherein said stimulating        compound comprises isoproterenol.        [103] The method of paragraph [98], wherein said endocrine cell        is an EC cell.        [104] The method of any one of paragraphs [86]-[103], wherein        one or more cells of said population of cells fails to localize        said selectable marker to a cell surface when contacted with        said stimulating compound.        [105] The method of paragraph [104], wherein said stimulating        compound is at least one of glucose or arginine and said one or        more cells is an EC cell.        [106] The method of paragraph [104], wherein said stimulating        compound is isoproterenol and said one or more cells is a β        cell.        [107] The method of any one of paragraphs [104]-[106], wherein        said selecting said target cell separates said target cell from        said one or more cells of said population of cells.        [108] A method comprising:    -   contacting a population of pancreatic progenitor cells or        precursors thereof with a composition comprising at least one        epigenetic modifying compound, wherein said contacting results        in an increased proportion of islet cells as compared to a        corresponding population of pancreatic progenitor cells which is        not contacted with said at least one epigenetic modifying        compound.        [109] The method of paragraph [108], wherein said islet cells        comprise at least one β cell.        [110] The method of paragraph [109], wherein said β cell        comprises an SC-β cell.        [111] The method of paragraph [110], wherein said SC-β cell        exhibits an in vitro glucose-stimulated insulin secretion        response to a glucose challenge.        [112] The method of paragraph [108], wherein said islet cells        comprise at least one alpha cell.        [113] The method of paragraph [108], wherein said islet cells        comprise a delta cell.        [114] The method of paragraph [108], wherein said islet cells        comprise a polyhormonal (PH) cell.        [115] The method of any one of paragraphs [108]-[114], further        comprising differentiating a plurality of stem cells in vitro to        obtain said population of pancreatic progenitor cells or        precursors thereof.        [116] The method of paragraph [115], wherein said stem cells are        human pluripotent stem cells.        [117] The method of any one of paragraphs [108]-[116], wherein        said at least one epigenetic modifying compound comprises one or        more of a DNA methylation inhibitor, a histone acetyltransferase        inhibitor, a histone deacetylase inhibitor, a histone        methyltransferase inhibitor, or a bromodomain inhibitor.        [118] The method of paragraph [117], wherein said at least one        epigenetic modifying compound comprises a histone        methyltransferase inhibitor.        [119] The method of paragraph [118], wherein said histone        methyltransferase inhibitor is an EZH2 inhibitor.        [120] The method of paragraph [118] or [119], wherein said        histone methyltransferase inhibitor is at least one of DZNep,        GSK126, or EPZ6438.        [121] The method of paragraph [120], wherein said histone        methyltransferase inhibitor is DZNep.        [122] The method of paragraph [121], wherein a concentration of        said DZNep in said composition is greater than 0.1 μM.        [123] The method of paragraph [122], wherein said concentration        of said DZNep is at least 0.5 μM.        [124] The method of paragraph [123], wherein said concentration        of said DZNep is about 1 μM.        [125] The method of any one of paragraphs [108] to [124],        wherein said at least one epigenetic modifying compound        comprises a histone deacetylase (HDAC) inhibitor.        [126] The method of paragraph [125], wherein said HDAC inhibitor        is a Class I HDAC inhibitor, a Class II HDAC inhibitor, or a        combination thereof.        [127] The method of paragraph [126], wherein said HDAC inhibitor        is at least one of KD5170, MC1568, or TMP195.        [128] The method of paragraph [127], wherein said HDAC inhibitor        is KD5170.        [129] The method of paragraph [108], wherein said at least one        epigenetic modifying compound comprises an HDAC inhibitor and an        EZH2 inhibitor.        [130] The method of paragraph [108], wherein said at least one        epigenetic modifying compound comprises DZNep and KD5170.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1. Histone Acetylation in hPSC-Derived Beta Cells

Systems approach (for instance Arda et al. (Cell Metabolism 23: 909-920,2016)) are used to identify age-dependent gene expression programs inhuman islet cells that includes (1) procurement of pancreatic tissuefrom children and adults, (2) developing robust and reliable cellpurification methods, (3) generation of comprehensive transcriptome andhistone modification maps, and (4) systematic assays of islet physiologyand function. Distinct modes of histone mediated regulation ofage-dependent genes in juvenile and adult human islet cells are revealedon the described genome wide histone map. Xu et al. (EMBO J 2014)describes that histone methylation is reduced in NGN3, NKX6.1, andNKX2.2 genes in endocrine progenitors. In NGN3 cells, H3K27me3 isdepleted from the NGN3, NeuroD1, and NKX6.1 elements, consistent withthe activation of the respective genes. Haumaitre et al. (MCB 2013)reports global reduction in HDAC expression and activity during pancreasdifferentiation. Xie (Cell Stem Cell, 2013) reports that chromatinarchitecture is inappropriately remodeled during in vitrodifferentiation. EZH2 inhibition and HDAC inhibition increases NGN3expression and leads to more stem cell derived beta cells, suggestingthat NGN3 is important for beta cell development.

Example 2. Inhibition of Histone Methylation and Deacetylation

The mRNA expression of NGN3 was investigated in each steps of directeddifferentiation described herein (FIG. 5 ), and it was observed thatcells expressed NGN3 to initiate SC-β cell differentiation (FIG. 7 ).The effects of inhibition of histone methylation and deacetylation onSC-β cell differentiation were investigated (FIG. 6 ). Stage 5 of thedirected differentiation as described herein (FIGS. 5-6 ) in hESC line(D97, D114, and D241 HUES8) was treated with an EZH2 inhibitor (DZNep),HDAC inhibitor (KD5170), and a combination thereof (FIG. 6 ).

Candidate inhibitor screening was performed as described in FIG. 18 .Inhibition of EZH2 or HDAC in stage 5 (D97 HUES8) increased endocrinecells and SC-β cells (FIG. 8 ). Combined inhibition of EZH2 and HDAC instage 5 (D114 HUES8) significantly increased endocrine cells (FIGS. 9-11). SC-β cells comprised 35.4% at the end of stage 5 with DZNep andKD5170. The total percentage of C-peptide positive cells was 39.6%,which suggested that there were at most 4% C-peptide positive, NKX6.1polyhormonal cells (FIG. 11 ). Combined inhibition of EZH2 and HDAC instage 5 was tested as described in FIG. 17 . Combined inhibition of EZH2and HDAC in stage 5 (D241 HUES8) increased Neorogenin3+ progenitors instage 5 (FIGS. 12-13 ). Combined inhibition of EZH2 and HDAC in stage 5increased NKX6.1+ progenitor cells (FIG. 16 ).

Inhibition with DZNep showed specific decrease in VMAT1+ INS− ECpopulation (FIG. 19 ), concomitant increase of NKX6.1-ChromA+ PH cells(FIG. 20 ), and unaffected NKX6.1+ INS+ SC-β cell population (FIG. 21 ).Inhibition with DZNep (1 μM) throughout stage 5 removed more than halfof the (VMAT1+ INS−) EC population without affecting viability (FIGS.19-21 ).

In summary, histone methyltransferase and deacetylase inhibitorsincreased NGN3 expression and beta cell differentiation, and EZH2inhibition by DZNep in stage 5 increased the proportion of endocrinecells (CHGA+), progenitors cells (NKX6-1+) and pre-SC-b cells(C-PEP+,NKX6-1+). Other EZH2 inhibitors (UNC1999, GSK126) did notincrease the proportion of all 3 cell types (FIG. 14 ). HDAC inhibitionby KD5170 in stage 5 increased the percentage of pre-SC-β cells (C-PEP+,NKX6-1+). Other HDAC inhibitors (sodium butyrate, TSA, SAHA) did notincrease the percentage of pre-SC-β cells (FIG. 15 ). Combined EZH2 andHDAC inhibition by DZNep and KD5170 in stage 5 (HUES8) synergisticallyand significantly enhanced differentiation of endocrine cells (CHGA+),progenitors cells (NKX6-1+) and pre-SC-b cells (C-PEP+,NKX6.1+).

Example 3. Differentiation of Stem Cell into Pancreatic β Cells (SC-βCells)

Two exemplary differentiation protocols v11 and v12, as illustrated inFIG. 39 , were tested. The two protocols are both 6-stage stepwiseprotocols that share similar reagents and their differentiation steps ofthe first 5 stages are largely based on the differentiation protocol asshown in FIG. 5 , and their differences are as follows: (a) in v12protocol, 0.25 μM DMH-1 was present in the culture medium on day 1 ofStage 3 (S3d1) and 20 ng/mL Activin A was present in the culture mediumon day 1 and day 2 of Stage 3 (S3d1-S3d2); (b) in v11 protocol there wasno DMH-1 or Activin A added in Stage 3; (c) in both vii and v12protocols, DZNep was present in the culture medium at 100 nM on day 1,3, 5, 7 at Stage 5. Stage 6 of both v11 and v12 protocols was carriedout by culturing product cells from Stage 5 in DMEM/F12 medium that issupplemented with 1% HAS without exogenous differentiation factors. Themedium was changed every other day throughout the Stage 6.

In one experiment, the cell cultures derived from stem cell line D705according to the two different protocols were examined at differentstages. FIG. 40 shows the flow cytometry results when surface expressionlevel of NKX6.1, Pdx1, Cdx2, ISL1, and CHGA were examined in thedifferentiated cells at Stage 4 (S4) and Stage 5 (S5), respectively. Asshown in the figure, the v12 protocol reduced the percentages of Cdx2+cells at S4 and ISL1−/NKX6.1− cells at S5, and increased the percentageof the differentiated ISL+/NKX6.1+ cells at S5.

The cell cultures were also examined after cryopreservation andreaggregation procedure (CryoRA cells) after Stage 5 and before recoveryof the cells for Stage 6 (S6) differentiation. In this experiment, assummarized in the graph in FIG. 41 , cell counting of the CryoRA cellclusters on day 11 of Stage 6 (S6d11) showed that the cell recoveryyield was much higher with the cells obtained via the v12 protocol ascompared to the cells obtained via the v11 protocol. Flow cytometryanalysis (FIG. 42 ) of the S6d11 cells obtained via the two protocolsindicated that the Sc-β cell composition constitutes comparablepercentage, about 40%. In vitro GSIS assay was also conducted to examinethe glucose response in S6d14 cells obtained via the two protocols,which showed comparable results between the two protocols. The insulincontent was also shown to be comparable (FIG. 43 ).

In one experiment, cell clusters comprising the pancreatic β cells weregenerated in bioreactors according to the exemplary protocol. In aglucose stimulated insulin secretion (GSIS) assay (FIG. 44A), SC-isletcell clusters were exposed sequentially to low (LG, 2.8 mM) or high (HG,20 mM) glucose conditions or a combination of 2.8 mM glucose and 30 mMKCl (KCl). An average of 6 independent batches is shown. SC-islet cellsclusters were also lysed and analyzed for total insulin content asanother method of analyzing activity (FIG. 44B). Data are Mean+/−SEM.

Example 4. SC-β Cell Surface Marker Identification Screen and Sorting

The cell surface marker library screen (Miltenyi MACS marker screen) foridentification of selective SC-β cell surface markers was performed with371 APC-conjugated monoclonal antibodies (FIGS. 22-23 ). The screeningshowed that 52.3% (194 out of 371) of wells contained sufficient cellsfor analysis. Live SC-β cell labeling with Newport green or fluozin-3and MACS marker screening can be performed as described in FIG. 24 .

Purification of SC-β cells by PSA-NCAM microbeads sorting was performedas described in FIG. 28 . PSA-NCAM micro-bead based sorting enrichedon-target cells and reduced SOX9+ cells (FIG. 25 ). EC cells (VMAT1+)remained after PSA-NCAM sorting (FIG. 26 ). The cells were cultured for14 days after sorting. PSA-NCAM expression decreased significantly uponEndo-N enzyme treatment (FIG. 27 ). Endo-N is an endosialidase whichdegrades rapidly and specifically linear polymers of sialic acid withα-2,8-linkage with a minimum length of 7-9 residues characteristic ofsialic acid residues associated with NCAM. Cleavage of PSA on NCAM inphysiological conditions.

To remove EC cells using PSA-NCAM, pre-exiting PSA-NCAM can be removedwith Endo-N enzyme, and PSA-NCAM can be selectively expressed on SC-βcells by stimulating with arginine. SC-β cells expressing PSA-NCAM canbe sorted using microbeads as described in FIG. 28 .

Example 5. Improving hESC Quality and DE Induction

Intestinal lineage is specified early in differentiation, and EC cellscan arise from the specified intestinal progenitors (FIG. 29 ). LowOct4% at stage 0 led to higher CDX2 percentage in later stages. HighOct4% was required for robust differentiation. Variability in Sox17induction remained even with high Oct4% (FIG. 30 ). CDX2+ intestinalpopulation dictated by stage 1 factor concentrations. Fine-tuningChir/AA concentration and timing can reduce CDX2 population and improveSC-β cell differentiation.

Example 6. Screening of EC Cell Differentiation Inhibitors

Further compound screening strategies on stage 5 cells to identifyinhibitors of EC cell differentiation are shown in FIGS. 31-32 .Matrigel coated 96 well plates are used to seed differentiated cells.Cells are plated at 0.1-0.3×10⁶ cells/well. For control, DMSO only isused. Wnt pathway library, epigenetics library, GPCR compound library,and stem cell signaling compound hormone library are screened atconcentrations 0.1 μM, 1.0 μM, and 10 μM in 0.1% DMSO. EC cellquantification is determined with high content image analysis with CDX2+as an early stage marker and VMAT+ as a late stage marker.

Example 7. γ-Irradiation of Stem Cell Derived Islet Cells

Proliferation of the population of stem cell derived islet cells wasinhibited with varying irradiation doses (8,000 rads and 10,000 rads).When no irradiation was performed, proliferation of cells andenlargement of the implant was observed (FIG. 33 ). High doseγ-irradiation (10,000 rads) had no significant impact on stem cellderived islet cell composition and function (FIG. 34 ). The ability tocontrol blood glucose was investigated for irradiated cryopreserved stemcell derived islet cells. The cryopreserved cells were thawed,irradiated, and implanted immediately after irradiation withoutrecovery. The cryopreserved stem cell derived islet cells lost abilityto control blood glucose 60 days after irradiation (FIG. 35 ). Enhancedbeta cell numbers was observed in irradiated sample (FIGS. 35-36 ). Inone experiment, γ-irradiation was conducted before thawing thecryopreserved stem cell derived islet cells, after which the cells wererecovered from cryopreservation and re-aggregated before being implantedinto animal models (Pre-thaw irradiation). As shown in FIG. 37 , ascompared to animals receiving implantation of stem cell derived isletcells that were irradiated after recovery from cryopreservation(Post-thaw irradiation), animals implanted with stem cell derived isletcells receiving Pre-thaw irradiation exhibited glycemic control for alonger period. All animals implanted with irradiated stem cell derivedislet cells showed glycemic control until the implant was explanted(FIG. 38 ).

In summary, the number of proliferating cells was dependent on dosage ofγ-irradiation. The number of proliferating cells was lower when the stemcell derived islet cells were irradiated with higher irradiation.γ-irradiation had no significant impact on composition and function ofthe stem cell derived islet cells. When γ-irradiation was conductedwhile the cells are frozen, the resultant stem cell derived islet cellimplants had longer glycemic control effect in the implanted animalmodels.

While preferred embodiments of the present disclosure have been shownand described herein, such embodiments are provided by way of exampleonly. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the disclosure. Itshould be understood that various alternatives to the embodiments of thedisclosure described herein can be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

What is claimed is:
 1. An in vitro population of cells derived from stemcells in vitro, wherein: a) at least 35% of the cells in the populationare NKX6.1-positive and ISL1-positive cells; b) at most 20% of the cellsin the population are NKX6.1-negative and ISL1-negative cells; c) atleast 60% of the cells in the population are CHGA-positive cells; and d)at least 30% of the cells in the population are C-peptide-positivecells.
 2. The population of cells of claim 1, wherein at least 40% ofthe cells in the population are NKX6.1-positive and ISL1-positive cells.3. The population of cells of claim 1, wherein at most 15% of the cellsin the population are NKX6.1-negative and ISL1-negative cells.
 4. Thepopulation of cells of claim 1, wherein at least 40% of the cells in thepopulation are C-peptide positive.
 5. The population of cells of claim1, wherein at least 50% of the cells in the population are C-peptidepositive.
 6. The population of cells of claim 1, wherein at least 55% ofthe cells in the population are C-peptide positive.
 7. The population ofcells of claim 1, wherein at least 70% of the cells in the populationare CHGA-positive cells.
 8. The population of cells of claim 1, whereinat least 80% of the cells in the population are CHGA-positive cells. 9.The population of cells of claim 1, wherein at least 85% of the cells inthe population are CHGA-positive cells.
 10. The population of cells ofclaim 1, wherein at least 40% of the cells in the population areNKX6.1-positive and C-peptide-positive cells.
 11. The population ofcells of claim 1, wherein: a) at least 35% of the cells in thepopulation are NKX6.1-positive and ISL1-positive cells; b) at most 15%of the cells in the population are NKX6.1-negative and ISL1-negativecells; c) at least 80% of the cells in the population are CHGA-positivecells; and d) at least 40% of the cells in the population areC-peptide-positive cells.
 12. The population of cells of claim 1,wherein: a) at least 40% of the cells in the population areNKX6.1-positive and ISL1-positive cells; b) at most 15% of the cells inthe population are NKX6.1-negative and ISL1-negative cells; c) at least85% of the cells in the population are CHGA-positive cells; and d) atleast 40% of the cells in the population are C-peptide-positive cells.13. The population of cells of claim 11, wherein at least 40% of thecells in the population are NKX6.1-positive and C-peptide-positivecells.
 14. The population of cells of claim 1, wherein the population ofcells are in one or more cell clusters.
 15. The population of cells ofclaim 11, wherein the population of cells are in one or more cellclusters.
 16. The population of cells of claim 1, wherein the populationcomprises cells that express NKX6.1 and ISL1 and that exhibit glucosestimulated insulin secretion (GSIS) in vitro.
 17. The population ofcells of claim 1, wherein the expression levels of NKX6.1, ISL1, CHGA,and C-peptide are determined utilizing flow cytometry.
 18. Thepopulation of cells of claim 1, wherein the population is in a devicesuitable for implantation into a human subject.
 19. The population ofcells of claim 11, wherein the population is in a device suitable forimplantation into a human subject.
 20. The population of cells of claim18, wherein the device comprises a semipermeable membrane.
 21. Thepopulation of cells of claim 19, wherein the device comprises asemipermeable membrane.